Nucleic acids for the identification of fungi and methods for using the same

ABSTRACT

Methods of detecting a dimorphic fungus, including differentiating a dimorphic fungus from other fungi are disclosed. A sample suspected of containing a nucleic acid of a fungus, such as an internal transcribed spacer-2 (ITS2) nucleic acid sequence of a dimorphic fungal rDNA, is screened for the presence or absence of that nucleic acid. The presence of the nucleic acid indicates the sample was contacted by the fungus. Determining whether the nucleic acid sequence is present in the sample can be accomplished by detecting hybridization between a dimorphic probe, species-specific probe, and/or microbe-specific probe and a nucleic acid sequence corresponding to the ITS2 region of fungal rDNA. Kits and arrays for carrying out these methods also are disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication No. 60/325,214, filed on Sep. 26, 2001.

FIELD

This invention relates to molecular identification of fungi, includingmethods of identifying fungi based on unique genetic characteristics,such as using nucleic acid probes to detect the presence of and identifynucleotides obtained from species and classes of fungi.

BACKGROUND

The incidence of disease caused by pathogenic and opportunistic fungihas been increasing over the past decade. See, e.g., McNeil, M. M, etal., Clin. Infect. Dis. 33:641-47 (2001); Ampel, N. M., et al., Clin.Infect. Dis. 27:1528-30 (1998); National Nosocomial InfectionsSurveillance (NNIS) System report, data summary from January 1990 to May1999, Am. J. Infect. Control 27:520-32 (1999). In humans, these fungalinfections are especially prevalent in people with suppressed immunesystems, such as HIV-positive and severely ill patients. For example,Penicillium marneffei is the third most common cause of opportunisticinfections in patients with AIDS in Thailand. Vanittanakom N.,Sirisanthana T., Curr. Top. Med. Mycol. 8:3542 (1997). Additionally, P.chrysogenum and P. citrinum have been recognized as the cause of humandisease.

Diagnosis of fungal infections is typically made by isolating theinfective organism in culture, by serologic assays, or throughhistopathologic examination of tissue. See, e.g., Hamilton, A. J., Med.Mycol. 36:351-64 (1998). However, pathogenic fungi may require severalweeks to grow, and a positive culture may represent benign colonization,rather than true invasion and infection, especially when opportunisticorganisms are isolated. Serologic tests on a single serum sample todetect circulating antifungal antibodies may be inconclusive (especiallyin immunosuppressed subjects). The acquisition of paired acute andconvalescent sera, which is necessary for a definitive serologicdiagnosis, requires an additional 3 to 4 weeks before convalescent serumcan be obtained. Morrison C. J., and Lindsley, M. D., in FungalPathogenesis: Principles and Clinical Applications (New York: MarcelDekker Inc., 2001; Calderone R. A., and Cihlar R. L., eds.). Therefore,histopathologic examination of tissue sections was often the most rapid,and sometimes the only, method available to diagnose invasive fungaldisease. However, histopathologic diagnosis of fungal infections isusually made through morphologic criteria, and fungi with atypicalmorphologic features can be difficult to identify and diagnose. Inaddition, fungi for which different anti-fungal therapies could be usedoften look morphologically similar in tissue sections.

The relatively recent development of automated DNA synthesis has allowedproduction of molecular probes with consistently defined properties thatmay result in increased test sensitivity, specificity, andreproducibility. Past research in the molecular identification of fungihas typically concentrated on a single species or genus of a fungus.See, e.g., LoBuglio, K. F., and J. W. Taylor, J. Clin. Microbiol.33:85-89 (1995); Loffler, J., et al., Med. Mycol. 36:275-79 (1998). Forexample, U.S. Pat. Nos. 5,631,132; 5,426,027; 5,635,353; and 5,645,992;and PCT publication WO 98/50584, disclose nucleic acid probes andmethods for detecting fungal species based on a certain region (the ITS2region) of rDNA. Additionally, some methods of molecular identificationof fungi can be very difficult or cumbersome to perform, or requireexpensive, specialized equipment. See, e.g., Sandhu, G. S., et al., J.Clin. Microbiol. 35:1894-96 (1997); Sandhu, G. S., et al., J. Clin.Microbiol. 33:2913-19 (1995); and Turenne, C. Y., et al., J. Clin.Microbiol. 37:1846-51 (1999).

SUMMARY

Disclosed is a method of detecting a dimorphic fungus, including amethod of differentiating the dimorphic fungus from non-dimorphic fungi,including fungi of the same biological genus.

In some embodiments, a sample suspected of containing a nucleic acid ofa fungus, such as an internal transcribed spacer-2 (ITS2) nucleic acidsequence of a dimorphic fungal rDNA, is screened for the presence orabsence of that nucleic acid. Any suitable sample, including abiological sample (e.g., blood, sputum, bronchoalveolar levage, orbiopsied tissue) can be used, and the nucleic acid can be amplifiedwithin the sample, such as by the polymerase chain reaction (PCR). Inparticular embodiments, the nucleic acid is detected and identified by apolymerase chain reaction enzyme-immunoassay (PCR-EIA). The presence ofthe nucleic acid indicates the sample was contacted by the fungus, suchas samples presently containing the fungus. The dimorphic fungi include,but are not limited to, Histoplasma capsulatum, Blastomycesdermatitidis, Coccidioides immitis, Paracoccidioides brasiliensis, andPenicillium marneffei.

If PCR is used to amplify the nucleic acid in the sample, an ITS1 orITS4 primer (listed in SEQ ID NO: 1 and SEQ ID NO: 2) can be used. Whilethe ITS3 sequence (SEQ ID NO: 3) can be used as a probe, ITS3 also canbe used as a PCR primer for amplifying the ITS2 region.

A sample can be prepared by processing and extracting nucleic acids froma sample. In some embodiments, a high-throughput DNA extractiontechnique is used to extract DNA from fungal cells. In particularembodiments, a mixture of plural types of glass microspheresdifferentiated by size (e.g., diameters of about 106 μm, about 0.5 mm;and about 3 mm) or differentiated according to a particular size ratio(e.g., a size ratio of a first microsphere to a second microsphere ofabout 1:5, a size ratio of the second microsphere to a third microsphereof about 1:6, and a size ratio of the first microsphere to the thirdmicrosphere of about 1:30) can be used. Alternatively, the ratio ofdiameters of the spheres is about 0.1 to 0.5 to 3.0.

Determining whether the nucleic acid sequence is present in the samplecan be accomplished by a variety of techniques. In some embodiments, aprobe is hybridized to an ITS2 nucleic acid, with detection ofhybridization indicating that the ITS2 nucleic acid is present in thesample (and that the sample came into contact with a dimorphic fungus).In particular embodiments, the probe comprises at least 15 contiguousnucleotides, such as at least 20 contiguous nucleotides, of thefollowing probe sequences: Dm (SEQ ID NO: 4), Hc (SEQ ID NO: 5), Bd (SEQID NO:6), Ci (SEQ ID NO: 7), Pb (SEQ ID NO: 8), or Pm (SEQ ID NO: 10).In more particular embodiments, the probe consists essentially of onethese sequences, such as Dm.

The probe can hybridize a segment of the ITS2 nucleic acid, such as aparticular half, third, quarter, or other subdivision of the ITS2region. In some embodiments, the probe hybridizes to a portion of theITS2 sequence beginning at about nucleotide 40 to 55, such as aboutnucleotide 44, 45, or 51, numbered after the end of the 5.8S codingsequence and extending downstream for the length of the probe.

In some embodiments, the method is capable of differentiating adimorphic fungus from a second dimorphic or non-dimorphic fungus, suchas Sporothrix schenckii, Cryptococcus neoformans, a Candida species, orPneumocystis carinii. In particular embodiments, the method is capableof differentiating Penicillium marneffei from Penicillium camembertii,Penicillium caseicolum, Penicillium chrysogenum, Penicillium glabrum,Penicillium griseofulvum, Penicillium italicum, Penicilliumjanthinellum, Penicillium purpurescens, Penicillium citrinum,Penicillium purpurogenum, Penicillium roquefortii, Penicilliumrubefaciens, Penicillium spinulosum, Sporothrix schenckii, Cryptococcusneoformans, a Candida species, an Aspergillus species, a Fusariumspecies, a Mucor species, a Rhizopus species, or Pneumocystis carinii.

Kits and arrays for carrying out these methods also are disclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B illustrate a generalized polymerase chainreaction-enzyme immunoassay (PCR-EIA) method.

FIG. 2 is a diagram of fungal rDNA, including the hybridization sites ofprimers and probes.

FIGS. 3A and 3B illustrate a sequence alignment indicating the dimorphicprobe (Dm) of several fungal species and the locations ofmicrobe-specific probes.

FIG. 4 is a digital image of PCR products amplified from DNA extractedfrom fungi using the disclosed high-throughput method and separated bysize on an agarose gel.

FIG. 5 is a digital image of an agarose gel demonstrating PCRsensitivity based on fungal genomic DNA isolated by the disclosed highthroughput method.

FIG. 6 is a digital image of an agarose gel demonstrating detection ofamplicons separated via gel electrophoresis.

FIG. 7 is a digital image of PCR products amplified from fungal DNA andseparated by size on an agarose gel.

SEQUENCE LISTING

The nucleic acid sequences listed in the accompanying sequence listingare shown using standard letter abbreviations for nucleotide bases. Onlyone strand of each nucleic acid sequence is shown, but the complementarystrand is understood as included by reference to the displayed strand.

SEQ ID NO: 1 shows the nucleic acid sequence of fungal universal forwardprimer ITS1.

SEQ ID NO: 2 shows the nucleic acid sequence of fungal universal reverseprimer ITS4.

SEQ ID NO: 3 shows the nucleic acid sequence of fungal universal captureprobe or forward primer ITS3.

SEQ ID NO: 4 shows the nucleic acid sequence of a probe for endemicdimorphic fungi.

SEQ ID NO: 5 shows the nucleic acid sequence of a probe to the ITS2region of Histoplasma capsulatum.

SEQ ID NO: 6 shows the nucleic acid sequence of a probe to the ITS2region of Blastomyces dermatitidis.

SEQ ID NO: 7 shows the nucleic acid sequence of a probe to the ITS2region of Coccidioides immitis.

SEQ ID NO: 8 shows the nucleic acid sequence of a probe to the ITS2region of Paracoccidioides brasiliensis.

SEQ ID NO: 9 shows the nucleic acid sequence of a probe to the ITS2region of Sporothrix schenckii.

SEQ ID NO: 10 shows the nucleic acid sequence of a probe to the ITS2region of Penicillium marneffei.

SEQ ID NO: 11 shows the nucleic acid sequence of a probe to the ITS2region of Cryptococcus neoformans.

SEQ ID NO: 12 shows the nucleic acid sequence of a probe to the ITS2region of Pneumocystis carinii.

SEQ ID NO: 13 shows the nucleic acid sequence of a probe to the ITS2region of Penicillium citrinum.

SEQ ID NO: 14 shows the nucleic acid sequence of a probe to the ITS2region of Penicillium purpurogenum.

DETAILED DESCRIPTION

Explanation of Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology canbe found in Benjamin Lewin, Genes VII, published by Oxford UniversityPress, 1999; Kendrew et al. (eds.), The Encyclopedia of MolecularBiology, published by Blackwell Science Ltd., 1994; and Robert A. Meyers(ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995; and other similarreferences.

As used herein, the singular forms “a,” “an,” and “the,” refer to boththe singular as well as plural, unless the context clearly indicatesotherwise. For example, the term “a probe” includes single or pluralprobes and can be considered equivalent to the phrase “at least oneprobe.”

As used herein, the term “comprises” means “includes.” Thus, “comprisinga dimorphic probe” means “including a dimorphic probe” without excludingother elements.

The term “or” refers to a single element of stated alternative elementsor a combination of two or more elements. For example, the phrase “theprobe comprising 15 contiguous nucleotides of SEQ ID NO: 4 or SEQ ID NO:10 refers to a probe comprising 15 contiguous nucleotides of SEQ ID NO:4, a probe comprising 15 contiguous nucleotides of SEQ ID NO: 10, or aprobe comprising 15 contiguous nucleotides of SEQ ID NO: 4 and 15contiguous nucleotides of SEQ ID NO: 10.

In order to facilitate review of the various embodiments of theinvention, the following explanations of terms are provided:

Amplification: of a nucleic acid molecule (e.g., a DNA or RNA molecule)refers to use of a technique that increases the number of copies of anucleic acid molecule in a sample. An example of amplification is thepolymerase chain reaction (PCR), in which a sample is contacted with apair of oligonucleotide primers under conditions that allow for thehybridization of the primers to a nucleic acid template in the sample.The primers are extended under suitable conditions, dissociated from thetemplate, re-annealed, extended, and dissociated to amplify the numberof copies of the nucleic acid. The product of amplification can becharacterized by such techniques as electrophoresis, restrictionendonuclease cleavage patterns, oligonucleotide hybridization orligation, and/or nucleic acid sequencing.

The amplification method can be modified in certain embodiments,including for example modification by additional steps or coupling theamplification with another protocol. For example, as described inExample 8, a polymerase chain reaction-enzyme immunoassay (PCR-EIA)method can be used for amplification and differentiation of fungi. Sucha PCR-EIA method is described in Elie, C. M., et al., J. Clin.Microbiol. 36:3260-65 (1998); and Fujita, S., et al., J. Clin.Microbiol. 33:962-67 (1995), and this PCR-EIA method can be modified tosuit particular embodiments.

FIGS. 1A-B illustrate a generalized PCR-EIA method. Denatured ampliconsare hybridized with a biotin-labeled capture probe (B) and adigoxigenin-labeled detection probe (D) in an Eppendorf® tube beforeaddition to wells of a streptavidin-coated microtiter plate (S).Horseradish peroxidase-conjugated anti-digoxigenin antibody is thenadded, and amplicons bound to the wells are detected colorimetrically atA_(650nm) after addition of TMB-H₂O₂ substrate.

Other examples of amplification include strand displacementamplification, as disclosed in U.S. Pat. No. 5,744,311;transcription-free isothermal amplification, as disclosed in U.S. Pat.No. 6,033,881; repair chain reaction amplification, as disclosed in WO90/01069; ligase chain reaction amplification, as disclosed inEP-A-320,308; gap filling ligase chain reaction amplification, asdisclosed in 5,427,930; and NASBA™ RNA transcription-free amplification,as disclosed in U.S. Pat. No. 6,025,134.

Animal: A living multi-cellular vertebrate or invertebrate organism, acategory that includes, for example, mammals and birds. The term mammalincludes both human and non-human mammals. Similarly, the term “subject”includes both human and veterinary subjects.

Sample: Encompasses a sample obtained from an animal, plant, or theenvironment. An “environmental sample” includes a sample obtained frominanimate objects or reservoirs within an indoor or outdoor environment.Environmental samples include, but are not limited to: soil, water,dust, and air samples; bulk samples, including building materials,furniture, and landfill contents; and other reservoir samples, such asanimal refuse, harvested grains, and foodstuffs.

A “biological sample” is a sample obtained from a plant or animalsubject. As used herein, biological samples include all clinical samplesuseful for detection of microbial or fungal infection in subjects,including, but not limited to, cells, tissues, and bodily fluids, suchas: blood; derivatives and fractions of blood, such as serum; extractedgalls; biopsied or surgically removed tissue, including tissues thatare, for example, unfixed, frozen, fixed in formalin and/or embedded inparaffin; tears; milk; skin scrapes; surface washings; urine; sputum;cerebrospinal fluid; prostate fluid; pus; bone marrow aspirates;bronchoalveolar levage (BAL); and saliva. In particular embodiments, thebiological sample is obtained from an animal subject, such as blood,serum, cerebrospinal fluid, BAL, pus, or prostate fluid.

cDNA (complementary DNA): A piece of DNA lacking internal, non-codingsegments (introns) and transcriptional regulatory sequences. cDNA alsocan contain untranslated regions (UTRs) that are responsible fortranslational control in the corresponding RNA molecule. cDNA can besynthesized in the laboratory by reverse transcription from messengerRNA extracted from cells.

Dimorphic: “Dimorphic,” or “thermally dimorphic,” describes a class offungi that demonstrate two different, temperature dependent morphologiesin their life cycles. At room temperature (about 25° C.), dimorphicfungi live in a mold-like phase, including growing hyphae. At bodytemperature (about 37° C.), dimorphic fungi demonstrate a yeast-likephase by forming yeast-like cells.

The endemic dimorphic fungi include, but are not limited to, thefollowing genera and species: Histoplasma capsulatum, Blastomycesdermatitidis, Coccidioides immitis, Paracoccidioides brasiliensis, andPenicillium marneffei. Other dimorphic fungi include, but are notlimited to, Sporothrix schenkii and Candida species.

Fungus: Living, single-celled and multicellular organisms belonging toKingdom Fungi. Most species are characterized by a lack of chlorophylland presence of chitinous cell walls, and some fungi can bemultinucleated. Representative, non-limiting examples of fungi includethe genera and species listed in Tables 2, 3, and 5-8 below, such asHistoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis,Paracoccidioides brasiliensis, Penicillium marneffei, Sporothrixschenckii, Cryptococcus neoformans, Candida species, Fusarium species,Rhizopus species, Aspergillus species, Mucor species, and Pneumocystiscarinii.

Homolog: A nucleotide sequence that shares a common ancestor withanother nucleotide sequence; the homologs diverged when a speciescarrying that ancestral sequence split into two species.

Isolated: An “isolated” microorganism (such as a bacteria, fungus, orprotozoan) has been substantially separated or purified away frommicroorganisms of different types, strains, or species. For example, acolony of Penicillium marneffei would be considered an “isolated”Penicillium marneffei. Microorganisms can be isolated by a variety oftechniques, including serial dilution and culturing.

An “isolated” biological component (such as a nucleic acid molecule,protein or organelle) has been substantially separated or purified awayfrom other biological components in the cell of the organism in whichthe component naturally occurs, i.e., other chromosomal andextra-chromosomal DNA and RNA, proteins, and organelles. Nucleic acidsand proteins that have been “isolated” include nucleic acids andproteins purified by standard purification methods. The term alsoembraces nucleic acids and proteins prepared by recombinant expressionin a host cell, as well as chemically synthesized nucleic acids.

ITS2: An internal transcribed spacer sequence of fungal rDNA. Asillustrated in FIG. 2, a diagram of the fungal rDNA region, the ITS2sequence is located between the 5.8S and 28S coding sequences.Additionally, hybridization sites for the ITS1 and ITS4 primers areshown in the phylogenetically conserved 18S and 28S rDNA regions, witharrows designating the direction of amplification (ITS1 is a forwardprimer, while ITS4 is a reverse primer). ITS3 (Biotin) represents thebiotinylated, universal fungal probe that binds in thephylogenetically-conserved, 5.8S rDNA region. However, non-biotynilatedITS3 also can be used as a forward primer. Probe (Digox.) representsdigoxigenin-labeled, species-specific probes which bind to the lesshighly conserved, ITS2 region, for example (and without limitation) theprobes listed in Table 1 below.

Oligonucleotide: A linear polynucleotide sequence of between 5 and 100nucleotide bases in length.

Operably linked: A first molecule, such as a nucleic acid or protein, isoperably linked with a second molecule when the first molecule is placedin a functional relationship with the second molecule. For instance, apromoter is operably linked to a nucleic acid coding sequence if thepromoter affects the transcription or expression of the coding sequence.Additionally, an intron is operably linked to an exon for the functionof splicing. Generally, operably linked nucleotide sequences arecontiguous and, where necessary to join two protein-coding regions, inthe same reading frame.

ORF (open reading frame): A series of nucleotide triplets (codons)coding for amino acids without any internal termination codons. Thesesequences are usually translatable into a peptide.

Probes and primers: Nucleic acid probes and primers can be readilyprepared based on the nucleic acid molecules provided in this invention.A probe comprises an isolated nucleic acid capable of hybridizing to atemplate nucleic acid, and a detectable label or reporter molecule canbe attached to a probe. Typical labels include radioactive isotopes,enzyme substrates, co-factors, ligands, chemiluminescent or fluorescentagents, haptens, and enzymes. Methods for labeling and guidance in thechoice of labels appropriate for various purposes are discussed in, forexample, Sambrook et al. (Molecular Cloning: A Laboratory Manual (ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001)) andAusubel et al., eds. (Short Protocols in Molecular Biology (John Wileyand Sons, New York, N.Y., 1999).

Primers are short nucleic acid molecules, for example DNAoligonucleotides 15 nucleotides or more in length. Primers can beannealed to a complementary target DNA strand by nucleic acidhybridization to form a hybrid between the primer and the target DNAstrand, and the primer can be extended along the target DNA strand by aDNA polymerase enzyme. Primer pairs can be used for amplification of anucleic acid sequence, e.g., by the polymerase chain reaction (PCR) orother nucleic acid amplification methods.

Methods for preparing and using probes and primers are described, forexample, in Sambrook et al.; Ausubel et al., eds.; and Innis et al. (PCRApplications, Protocols for Functional Genomics (Academic Press, Inc.,San Diego, Calif., 1999)). PCR primer pairs can be derived from a knownsequence, for example, by using computer programs intended for thatpurpose such as Primer3 (Whitehead Institute for Biomedical Research,Cambridge, Mass.; this program is accessible through the WhiteheadInstitute's website).

The specificity of a particular probe or primer increases with itslength. Thus, as one non-limiting example, a primer comprising 15consecutive nucleotides of the P. marneffei ITS2 sequence will anneal toa target sequence, such as another ITS2 homolog from the familycontained within a P. marneffei genomic DNA library, with a higherspecificity than a corresponding primer of only 10 nucleotides; thus, inorder to obtain greater specificity, probes and primers can be selectedthat comprise 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or moreconsecutive nucleotides of P. marneffei ITS2 sequences.

Included are isolated nucleic acid molecules (such as probes andprimers) that comprise specified lengths of a fungal ITS2 sequence. Suchmolecules can comprise at least 10, 15, 20, 25, 30, 35, 40, 50, 75, 100,or more consecutive nucleotides of the ITS2 sequence, and can beobtained from any region of the ITS2 sequence. In some embodiments, theITS2 region is subdivided into smaller parts, such as halves, thirds,quarters, fifths, sixths, tenths, or twentieths. In particularembodiments, these divided portions are numbered sequentially beginningwith the portion adjacent the 5.8S coding region. By way of exampleonly, an ITS2 sequence can be apportioned into halves, thirds, orquarters based on sequence length, and the isolated nucleic acidmolecules can be derived from the first or second halves of themolecules, any of the three thirds of the molecules, or any of the fourquarters of the molecules.

The generalized ITS2 sequence is diagrammed in FIG. 2, and ITS2sequences for particular fungi can be cloned and sequenced usingstandard techniques (e.g., as described in Sambrook et al.) or obtainedfrom public or private sequence databases, such as GenBank. Particularsequences are listed in Table 1 and the accompanying sequence listing.

The fungal sequence amplified by the ITS1 and ITS4 primers is generallyabout 600 nucleotides in length and can be hypothetically divided intoabout halves (e.g., nucleotides 1 to about 300 and about 301 to about600) or about quarters (e.g., nucleotides 1 to about 150, about 151 toabout 300, about 301 to about 450, and about 451 to about 600), forinstance. However, as stated above, other divisions into segments arepossible, such as tenths, fifths, sixths, and eighths. Additionally, anyparticular portion of the ITS2 region can be identified with referenceto particular nucleotides, for example (and without limitation)nucleotides 1-55, 40-95, 70-130, 83-157, 200-425, or 315 to 600.

As illustrated in FIG. 2, the ITS2 region is located between the 5.8Sand 28S coding sequences of fungal rDNA. The ITS2 region is about 200nucleotides long, and the nucleotides in the ITS2 sequence can benumbered beginning with the first nucleotide downstream of the 5.8Scoding sequence and ending with the last nucleotide directly in advanceof the 28S coding sequence. Similar to the entire ITS1-ITS4 amplicon,the ITS2 region can be divided into segments, for example (and withoutlimitation), halves, thirds, quarters, fifths, or tenths.

While the length of the ITS2 sequence is about 200 nucleotides in fungi,the sequence can be shorter or longer in a particular fungal species,thus altering the nucleotide reference numbers for a particular species.For example, an ITS2 sequence from a particular species that is about180 nucleotides in length could be split in half by nucleotides 1-90 and91-180, or quarters by nucleotides 1-45, 46-90, 91-135, and 136-180.

Segments of an ITS 1-ITS4 amplicon or an ITS2 region can include one ormore nucleotides that overlap into a different a segment (half, quarter,third, tenth, etc.), so long as the majority of the sequence lies withina particular segment. For example, a 25-nucleotide probe or primercorresponding to nucleotides 135 to 160 of a 600 nucleotide ITS1-ITS4amplicon is considered to fall within the first quarter of the sequence,even though 10 nucleotides overlap into the second quarter.

Nucleic acid molecules can be selected that comprise at least 10, 15,20, 25, 30, 35, 40, 50, 75, 100, or more consecutive nucleotides of anyof these portions of the fungal ITS2 sequence. Furthermore, nucleic acidmolecules can include only a portion of a particular probe or primer,such as a sequence of 5, 10, 15, 20, 25, 30, 40, 50, or more contiguousnucleotides of a particular primer, including (without limitation) theprobes and primers disclosed herein.

Primers include the universal fungal primers for PCR amplificationcommonly known as ITS 1, ITS3, ITS4. These primers are listed in Table1, illustrated in FIG. 2, and their sequences are provided by SEQ IDNOS: 1, 2, and 3.

As one non-limiting example, the Dm probe can comprise at least 25consecutive nucleotides of the first half (nucleotides 1-100), firstthird (nucleotides 1-66), first quarter (nucleotides 1-50), second third(nucleotides 67-132), or second quarter (nucleotides 50-100) of an ITS2region 200 nucleotides in length. Sequences from particular fungicorresponding to Dm are illustrated in FIG. 3. In this figure, the Dmsequence is indicated by the shaded box in the upper portion of FIG. 3A;sequences are compared to that of Histoplasma capsulatum, with identicalbases indicated by a period (.) and a gap in the sequence indicated by adash (-). The Dm sequence begins at about nucleotide 30 to aboutnucleotide 60 of the ITS2 sequence, depending on the species of fungus,for example (and without limitation) at nucleotide 51 in Histoplasmacapsulatum, nucleotide 45 in Blastomyces dermatitidis, nucleotide 51 inCoccidioides immitis, nucleotide 45 in Paracoccidioides brasiliensis,and nucleotide 44 in Penicillium marneffei. The Dm probe includes theparticular nucleotide sequence from Histoplasma capsulatum and similarsequences, such as sequences demonstrating about 55% to about 100%sequence identity, such as about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 99% or greater sequence identity, including the particularsequences of the fungi illustrated in FIGS. 3A-B.

As another, non-limiting example, the Pm probe—a species-specific probeto P. marneffei—can comprise at least 18 consecutive nucleotides of theportion consisting of nucleotides 90-140 of the P. marneffei ITS2sequence. Other species-specific and microbe-specific probes areillustrated by shaded boxes in FIG. 3A-B.

A “species-specific” probe is a probe shown to be capable ofdifferentiating a species of fungus from another species in the samegenus. A “microbe-specific” probe is a probe that is capable ofdifferentiating a species of fungus from another species in a differentgenus, but that has not yet been shown to be capable of differentiatingthat species of fungus from another species in the same genus. However,if a microbe-specific probe is shown to be capable of differentiatingfungi within the same genus, then that probe can properly be describedas “species specific.” For example, the Bd probe is considered to be amicrobe-specific probe capable of differentiating B. dermatitidis fromother fungi. Because B. dermatitidis is the only currently known specieswithin the Blastomyces genus, it cannot currently be determined whetherthe Bd probe also is a species-specific probe. Another example of amicrobe-specific probe would be a probe capable of differentiatingPenicillium species from Candida species, which can be described as a“genus-specific” probe.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination can be accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, e.g., by genetic engineering techniques.

Sequence identity: The similarity between two nucleic acid sequences isexpressed in terms of the similarity between the sequences, otherwisereferred to as sequence identity. Sequence identity is frequentlymeasured in terms of percentage identity, similarity, or homology; ahigher percentage identity indicates a higher degree of sequencesimilarity. Homologs of fungal ITS2 sequences will possess a relativelyhigh degree of sequence identity when aligned. Typically, fungal ITS2sequences are about 80 to 100% identical at the nucleotide level whencomparing homologs of the same species. However, within particularportions of the IFS2 region, the sequence identity can be more or less.For example, among the dimorphic fungi, the portion of the ITS2 regionadjacent to the 5.8S coding sequence demonstrates greater sequenceidentity (such as about 84 to 100%), while the portion of the ITS2region adjacent the 28S sequence demonstrates lesser sequence identity(such as about 0 to 44%).

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al.(1990) J. Mol. Biol. 215:403-410) is available from several sources,including the National Center for Biotechnology Information (NCBI,Bethesda, Md.) and on the Internet, for use in connection with thesequence analysis programs blastp, blastn, blastx, tblastn and tblastx.It can be accessed through the NCBI website. A description of how todetermine sequence identity using this program also is available on theNCBI website.

When less than the entire sequence is being compared for sequenceidentity, homologs typically possess at least 75% sequence identity overshort windows of 10-20 amino acids, and can possess sequence identitiesof at least 85%, or at least 90%, or even 95% or greater, depending ontheir similarity to the reference sequence. Methods for determiningsequence identity over such short windows are described on the NCBIwebsite.

These sequence identity ranges are provided for guidance only; it isentirely possible that strongly significant homologs could be obtainedthat fall outside of the ranges provided.

An alternative indication that two nucleic acid molecules are closelyrelated is that the two molecules hybridize to each other understringent conditions. Stringent conditions are sequence-dependent andare different under different environmental parameters. Generally,stringent conditions are selected to be about 5° C. to 20° C. lower thanthe thermal melting point (Tm) for the specific sequence at a definedionic strength and pH. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of the target sequence hybridizes to aperfectly matched probe. Conditions for nucleic acid hybridization andcalculation of stringencies can be found in Sambrook et al. and P.Tijssen, Hybridization With Nucleic Acid Probes, Part I: Theory andNucleic Acid Preparation (Laboratory Techniques in Biochemistry andMolecular Biology) (Elsevier Science Ltd., NY, N.Y., 1993). Nucleic acidmolecules that hybridize under stringent conditions to a fungal ITS2sequence will typically hybridize to a probe based on either an entireITS2 sequence or selected portions of the ITS2 sequence under washconditions of 2×SSC at 50° C.

For purposes of the present disclosure, “stringent conditions” encompassconditions under which hybridization will only occur if there is lessthan 25% mismatch between the hybridization molecule and the targetsequence. “Stringent conditions” may be broken down into particularlevels of stringency for more precise definition. Thus, as used herein,“moderate stringency” conditions are those under which molecules withmore than 25% sequence mismatch will not hybridize; conditions of“medium stringency” are those under which molecules with more than 15%mismatch will not hybridize; and conditions of “high stringency” arethose under which sequences with more than 10% mismatch will nothybridize. Conditions of “very high stringency” are those under whichsequences with more than 6% mismatch will not hybridize.

Transformed: A transformed cell is a cell into which a nucleic acid hasbeen introduced by molecular biology techniques. The term“transformation” encompasses all techniques by which a nucleic acidmolecule might be introduced into such a cell, including transfectionwith viral vectors, transformation with plasmid vectors, andintroduction of naked DNA by such techniques as electroporation,lipofection, and particle gun acceleration.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector can include nucleic acidsequences that permit it to replicate in a host cell, such as an originof replication. A vector also can include one or more selectable markernucleic acids and other genetic elements.

High Throughput Isolation of Fungal DNA

Disclosed is a high-throughput method for isolating DNA from prokaryoticand eukaryotic cells, including cells of bacteria, fungi, protozoans,animals, and plants. Cells are mechanically disrupted using pluralgranular materials, such as beads or microspheres, and standardlaboratory equipment. For example, the mechanical energy for disruptingcells can be supplied by a rotary incubator (such as models availablefrom New Brunswick Scientific, Edison, N.J.), rather than specializedequipment. After cell disruption, cellular proteins are precipitated,such as by using a concentrated salt solution, and the samples aretreated with RNase. The remaining nucleic acids (i.e., DNA) arecollected from the sample, such as by spin columns or precipitation byalcohol, followed by centrifugation.

The granular materials can be composed of any substance, such as metal,polymer, or silica, and the individual grains can be of any shape, forexample (but not limited to) spherical, cubical, or pyramidal. Someembodiments employ glass microspheres.

The grains can be of any suitable size. In some embodiments, the grainsare about 10 μm to 5 mm, such as about 50 μm to 4 mm, or, in particularembodiments, about 100 μm to about 3 mm.

The plural granular materials can be differentiated by type of materialor size of grains. In some embodiments, glass microspheres of varyingsizes, such as about 100 μm to about 3 mm, are used. In particularembodiments, three different sizes of glass microspheres are used: afirst set of glass microspheres of about 100 μm, such as 106 μm; asecond set of glass microspheres of about 0.5 mm; and a third set ofglass microspheres of about 3 mm. In other particular embodiments, themicrospheres can differ in diameter by a particular ratio, such as about1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:30, 1:50,1:100, 1:500, or any other ratio within this range. For example, andwithout limitation, if three differently sized microspheres are used'A,B, and C—the A:B diameter ratio can be about 1:5, the B:C diameter ratiocan be about 1:6, and the A:C diameter ratio can be about 1:30.Alternatively, the ratio of diameters can be about X:Y:Z, where X isabout 0.05 to 0.2, Y is about 0.3 to 0.7, and Z is about 2.5 to 3.5. Inparticular embodiments, the ratio is about 0.1 to 0.5 to 3.0.

In still other particular embodiments, the microspheres can be selectedaccording to a desired size difference. For example, and withoutlimitation, a diameter difference of about (or at least about) 100 μm,200 μm, 500 μm, 1 mm, 2 mm, or 5 mm can be provided.

Multiple biological samples can be processed together via thishigh-throughput method. Samples can be arranged in an array, such as amulti-well plate. Each well can contain a sample from the same ordifferent source, and plates with different numbers of wells can beused, such as an 18-well, 24-well, 36-well, 48-well, 72-well, 96-well,or 120-well plate. Plates having greater numbers of wells can be used,and the individual wells can be of any suitable volume, such as (andwithout limitation) about 0.5 ml, 1.0 ml, or 2.0 ml. In particularembodiments, a 96-well plate having 2.0 ml wells is used.

One particular, non-limiting example of this high-throughput method isprovided in Example 2.

Probes

Probes capable of hybridizing to isolated fungal rDNA are disclosed,some of which are species-specific or microbe-specific. Additionally,probes that differentiate dimorphic fungi (which, in turn, can bemicrobe-specific or species-specific from other yeast-like fungi, oryeast species, are disclosed and referred to herein as “dimorphicprobes.” The species-specific, microbe-specific, and dimorphic probesinclude sequences obtained from the ITS2 sequence of fungal DNA.

One particular dimorphic probe, Dm, is described in Table 1 and FIG. 3.Other, exemplary, non-limiting, probes include the species-specific andmicrobe-specific probes listed in Tables 1-3.

In some embodiments, the species-specific probes correspond to sequencesin the downstream half, third, quarter, fifth, sixth, or tenth of theITS2, such as sequences adjacent to the 28S coding sequence, while thedimorphic probes correspond to sequences in the upstream half, third,quarter, fifth, sixth, or tenth of the ITS2, such as sequences adjacentto the 5.8S coding sequence. However, in some embodiments, a minorportion of the probe sequence corresponds to sequences that fall withinthe 5.8S or 28S coding sequences. Particular dimorphic probes correspondto sequences of about 15 to about 50 nucleotides, such as about 25nucleotides, within the portion of the ITS2 region from about nucleotide40 to about nucleotide 80, while particular microbe-specific probescorrespond to sequences of about 10 to about 50 nucleotides, such asabout 15 to about 30 nucleotides, within the portion of the ITS2 regionfrom about nucleotide 90 to about nucleotide 200.

Detecting Fungal rDNA Sequences

The presence of a fungus within a sample can be detected using the probeand primer sequences described herein. Fungal DNA can be directlydetected or amplified prior to detection, and identification of thefungi from which the DNA originated can be confirmed byspecies-specific, microbe-specific, and/or dimorphic oligonucleotideprobes. The methods described herein can be used for any purpose wherethe detection of fungi is desirable, including diagnostic and prognosticapplications, such as in laboratory and clinical settings.

Appropriate samples include any conventional environmental or biologicalsamples, including clinical samples obtained from a human or veterinarysubject, for instance blood or blood-fractions (e.g., serum), sputum,saliva, oral washings, skin scrapes, biopsied tissue, BAL, cerebrospinalfluid, or prostate fluid. Standard techniques for acquisition of suchsamples are available. See, e.g. Schluger et al., J. Exp. Med.176:1327-33 (1992); Bigby et al., Am. Rev. Respir. Dis. 133:515-18(1986); Kovacs et al., NEJM 318:589-93 (1988); and Ognibene et al., Am.Rev. Respir. Dis. 129:929-32 (1984). Serum or other blood fractions canbe prepared according to standard techniques; about 200 μL of serum isan appropriate amount for the extraction of DNA for use in amplificationreactions. See, e.g., Schluger et al.; Ortona et al., Mol. Cell Probes10:187-90 (1996).

The sample can be used directly or can be processed, such as by addingsolvents, preservatives, buffers, or other compounds or substances.

Once a sample has been obtained, DNA can be extracted using standardmethods. For instance, rapid DNA preparation can be performed using acommercially available kit (e.g., the InstaGene Matrix, BioRad,Hercules, Calif.; the NucliSens isolation kit, Organon Teknika,Netherlands; the QIAGEN Tissue Kit, QIAGEN, Inc., Valencia, Calif.). TheDNA preparation technique can be chosen to yield a nucleotidepreparation that is accessible to and amenable to nucleic acidamplification. Particular DNA extraction and preparation methods include(without limitation) the high-throughput method described above and themethod described in Example 1 below.

Fungal nucleotide sequences can be detected through the hybridization ofan oligonucleotide probe to nucleic acid molecules extracted from abiological or environmental sample, including a clinical sample. Thesequence of appropriate oligonucleotide probes will correspond to aregion within one or more of the fungal nucleotide sequences disclosedherein. Standard techniques can be used to hybridize fungaloligonucleotide probes to target sequences, such as the techniquesdescribed in U.S. Pat. Nos. 5,631,132; 5,426,027; 5,635,353; and5,645,992; and PCT publication WO 98/50584.

In some embodiments, the probe is detectably labeled in some fashion,either with an isotopic or non-isotopic label; in alternativeembodiments, the target (template) nucleic acid is labeled. Non-isotopiclabels can, for instance, comprise a fluorescent or luminescentmolecule, or an enzyme, co-factor, enzyme substrate, or hapten. Theprobe is incubated with a single-stranded preparation of DNA, RNA, or amixture of both, and hybridization determined after separation of doubleand single-stranded molecules. Alternatively, probes can be incubatedwith a nucleotide preparation after it has been separated by size and/orcharge and immobilized on an appropriate medium.

In some embodiments, target fungal nucleotide sequences in a sample areamplified prior to using a hybridization probe for detection. Forinstance, it can be advantageous to amplify part or all of the ITS2sequence, then detect the presence of the amplified sequence pool. Anynucleic acid amplification method can be used, including the polymerasechain reaction (PCR) amplification. In particular embodiments, thePCR-EIA method is used for the amplification and detection of fungi; thePCR-EIA is described herein, such as in Example 8, and illustrated inFIGS. 1-2.

The sequential use of universal fungal primers for PCR amplification andmicrobe-specific probes can be used to identify fungi. Universal fungalprimers directed to the ITS1 and ITS4 regions of rDNA (See FIG. 2) allowamplification of a major portion of rDNA from most fungi, rather thanthat from only a single fungal species. The rDNA gene offers a suitableamplification target, not only because it contains binding sites foruniversal fungal primers, but also because the chromosome on which thisgene is located contains approximately 100 gene copies that offer“pre-amplification” to increase amplicon yield and test sensitivity.Therefore, in some embodiments, the use of universal primers and amultiple copy gene target (rDNA) has greater utility and sensitivity forthe identification of fungi in diverse samples than offered by genetargets of other embodiments.

While PCR-EIA offers one method of amplicon detection and identificationof fungi, other methods of identification can be used. Amplicons can beproduced using microbe-specific primers and detected by electrophoresisin agarose gels and ethidium bromide staining. See, e.g., Aoki, F. H.,et al., J. Clin. Microbiol. 37:315-20 (1999). The presence of a band insuch an agarose gel is considered a positive result using specificprimers. However, because different species of fungi can produce similarsize amplicons, amplicon size detection can be supplemented with otheridentification methods.

Alternatively, the presence of a unique banding pattern afterrestriction enzyme digestion of fungal DNA, including DNA obtained byamplification, such as PCR, can be used for species identification,including methods commonly known as “genetic fingerprinting” based onrestriction-fragment length polymorphism (RFLP) or randomly amplifiedpolymorphic DNA (RAPD) analysis. See, e.g., Morace, G., M. et al., J.Clin. Microbiol. 35:667-72 (1997).

Species-specific probes can be used to obtain a final identification ofa fingus using Southern blot, slot blot, dot blot, or another similarmethod. See, e.g., Sandhu, G. S., et al., J. Clin. Microbiol. 35:1894-96(1997); Sandhu, G. S., et al., J. Clin. Microbiol. 33:2913-19 (1995);and Tanaka, K., et al., J. Clin. Microbiol. 34:2826-28 (1996).Additionally, an identification method using universal primers wasdeveloped by Turenne et al. (J. Clin. Microbiol. 37:1846-51 (1999)), inwhich fungi are identified by the exact size of amplified DNA using anautomated fluorescent capillary electrophoresis system.

The probes described herein not only provide a means to identify fungiin culture, but also aid in the histological identification of fungi inother samples, such as environmental and biological samples. Applicationof these probes to fungi in tissue sections can allow thedifferentiation of truly invasive organisms from simple colonizers, andmultiple techniques can be employed to identify fungi in tissue usingthese probes. In some embodiments, fungal DNA is extracted from thetissue and identified by PCR-EIA. In other embodiments, the probes canbe used for in situ hybridization, allowing localization of fungal DNAdirectly in the tissue. In still other embodiments, the combination ofPCR and in situ hybridization procedures, where the target DNA is bothamplified and hybridized in situ, can be employed. None of these methodsshould be considered mutually exclusive, however.

PCR-EIA allows amplification and detection of small quantities of DNA,such as quantities of only a few nanograms, a few picograms, or less.Conceivably, PCR-EIA can be able to detect only a few molecules offungal DNA present in a sample. However, sensitivity of a PCR-basedassay, such as PCR-EIA, can be enhanced by various modifications of thetechnique. For example, nested PCR utilizes a second set of primers,internal to the original set of primers, to re-amplify the target DNAusing the amplicons from the first PCR as a template for the second PCR.See, e.g., Podzorski, R. P., and D. H. Persing, in Manual of ClinicalMicrobiology, 6th ed., P. R. Murray, et al. (eds.), (ASM Press,Washington, DC, 1995); and Rappelli, P., R., et al., J. Clin. Microbiol.36:3438-40 (1998). Additionally, the PCR reaction can be continuedthrough more cycles, continuing the geometric increase of DNA amplified,and alternative forms of Taq polymerase are available that haveincreased stability and accuracy throughout an increased number of PCRcycles. Commercially available Taq polymerases can be obtained fromRoche Molecular Systems (Pleasanton, Calif.), Seikagaku America(Falmouth, Conn.), and other commercial suppliers.

Fungal Profiling Arrays

An array containing a plurality of heterogeneous, dimorphic,microbe-specific, and/or species-specific probes can be used to screen asample for the presence of a fungus. Such arrays can be used to rapidlydetect and identify a fungus, for example a dimorphic fungus or a fungusof a particular species or genus, such as Penicillium marneffei.

Arrays are arrangements of addressable locations on a substrate, witheach address containing a nucleic acid, such as a probe. In someembodiments, each address corresponds to a single type or class ofnucleic acid, such as a single probe, though a particular nucleic acidcan be redundantly contained at multiple addresses. A “microarray” is aminiaturized array requiring microscopic or otherwise assistedexamination for detection of hybridization. Larger “macroarrays” alloweach address to be recognizable by the naked human eye and, in someembodiments, a hybridization signal is detectable without additionalmagnification. The addresses can be labeled, keyed to a separate guide,or otherwise identified by location.

In some embodiments, a fungal profile array is a collection of separateprobes at the array addresses. As one, non-limiting example, the arraycan contain the probes listed in Table 1. The fungal profiling array isthen contacted with a sample suspected of containing fungal nucleicacids under conditions allowing hybridization between the probe andnucleic acids in the sample to occur. Any sample potentially containing,or even suspected of containing, fungal nucleic acids can be used,including nucleic acid extracts, such as amplified or non-amplified DNAor RNA preparations. A hybridization signal from an individual addresson the array indicates that the probe hybridizes to a nucleotide withinthe sample. This system permits the simultaneous analysis of a sample byplural probes and yields information identifying the fungal DNA or RNAcontained within the sample. In alternative embodiments, the arraycontains fungal DNA or RNA and the array is contacted with a samplecontaining a probe. In any such embodiment, either the probe or thefungal DNA or RNA can be labeled to facilitate detection ofhybridization.

The nucleic acids can be added to an array substrate in dry or liquidform. Other compounds or substances can be added to the array as well,such as buffers, stabilizers, reagents for detecting hybridizationsignal, emulsifying agents, or preservatives.

Within an array, each arrayed nucleic acid is addressable—its locationcan be reliably and consistently determined within the at least the twodimensions of the array surface. Thus, ordered arrays allow assignmentof the location of each nucleic acid at the time it is placed within thearray. Usually, an array map or key is provided to correlate eachaddress with the appropriate nucleic acid. Ordered arrays are oftenarranged in a symmetrical grid pattern, but nucleic acids could bearranged in other patterns (e.g., in radially distributed lines, a“spokes and wheel” pattern, or ordered clusters).

An address within the array can be of any suitable shape and size. Insome embodiments, the nucleic acids are suspended in a liquid medium andcontained within square or rectangular wells on the array substrate.However, the nucleic acids can be contained in regions that areessentially triangular, oval, circular, or irregular. The overall shapeof the array itself also can vary, though in some embodiments it issubstantially flat and rectangular or square in shape.

Fungal profiling arrays can vary in structure, composition, and intendedfunctionality, and can be based on either a macroarray or a microarrayformat, or a combination thereof. Such arrays can include, for example,at least 10, at least 25, at least 50, at least 100, or more addresses,usually with a single type of nucleic acid at each address. In the caseof macroarrays, sophisticated equipment is usually not required todetect a hybridization signal on the array, though quantification can beassisted by standard scanning and/or quantification techniques andequipment. Thus, macroarray analysis as described herein can be carriedout in most hospitals, agricultural and medial research laboratories,universities, or other institutions without the need for investment inspecialized and expensive reading equipment.

Examples of substrates for the phage arrays disclosed herein includeglass (e.g., functionalized glass), Si, Ge, GaAs, GaP, SiO₂, SiN₄,modified silicon nitrocellulose, polyvinylidene fluoride, polystyrene,polytetrafluoroethylene, polycarbonate, nylon, fiber, or combinationsthereof. Array substrates can be stiff and relatively inflexible (e.g.,glass or a supported membrane) or flexible (such as a polymer membrane).One commercially available product line suitable for probe arraysdescribed herein is the Microlite line of Microtiter® plates availablefrom Dynex Technologies UK (Middlesex, United Kingdom), such as theMicrolite 1+ 96-well plate, or the 384 Microlite+ 384-well plate.

Addresses on the array should be discrete, in that hybridization signalsfrom individual addresses should be distinguishable from signals ofneighboring addresses, either by the naked eye (macroarrays) or byscanning or reading by a piece of equipment or with the assistance of amicroscope (microarrays).

Addresses in a macroarray can be of a relatively large size, such aslarge enough to permit detection of a hybridization signal without theassistance of a microscope or other equipment. Thus, addresses of amacroarray can be as small as about 0.1 mm across, with a separation ofabout the same distance. Alternatively, addresses can be about 0.5, 1,2, 3, 5, 7, or 10 mm across, with a separation of a similar or differentdistance. Larger addresses (larger than 10 mm across) are employed incertain embodiments. The overall size of the array is generallycorrelated with size of the addresses (i.e., larger addresses willusually be found on larger arrays, while smaller addresses can be foundon smaller arrays). Such a correlation is not necessary, however.

The arrays herein can be described by their densities—the number ofaddresses in a certain specified surface area. For macroarrays, arraydensity can be about one address per square decimeter (or one address ina 10 cm by 10 cm region of the array substrate) to about 50 addressesper square centimeter (50 targets within a 1 cm by 1 cm region of thesubstrate). For microarrays, array density will usually be one or moreaddresses per square centimeter, for instance, about 50, about 100,about 200, about 300, about 400, about 500, about 1000, about 1500,about 2,500, or more addresses per square centimeter.

The use of the term “array” includes the arrays found in DNA microchiptechnology. As one, non-limiting example, the probes could be containedon a DNA microchip similar to the GeneChip® products and relatedproducts commercially available from Affymetrix, Inc. (Santa Clara,Calif.). Briefly, a DNA microchip is a miniaturized, high-density arrayof probes on a glass wafer substrate. Particular probes are selected,and photolithographic masks are designed for use in a process based onsolid-phase chemical synthesis and photolithographic fabricationtechniques, similar to those used in the semiconductor industry. Themasks are used to isolate chip exposure sites, and probes are chemicallysynthesized at these sites, with each probe in an identified locationwithin the array. After fabrication, the array is ready forhybridization. The probe or the nucleic acid within the sample can belabeled, such as with a fluorescent label and, after hybridization, thehybridization signals can be detected and analyzed.

Detecting Infection and Disease

The method also includes diagnosing or detecting certain classes ofinfections with dimorphic fungi, by using the Dm probe (or a fragment orvariant thereof) to detect the presence of infection with a dimorphicfungus, such as H. capsulatum, B. dermatitidis, C. immitis, P.brasiliensis, or P. marneffei. Once the presence of the dimorphic fungalinfection is established using the Dm probe, these different dimorphicfungal infections can be further distinguished from one another byexposing the specimen to a microbe-specific or species-specific probe,such as the Hc probe (or a fragment or variant thereof) whichspecifically binds to H. capsulatum but not B. dermatitidis, C. immitis,P. brasiliensis, or P. marneffei; the Bd probe which binds specificallyto B. dermatitidis but not H. capsulatum, C. immitis, P. brasiliensis,or P. marneffei; the Ci probe (or a variant or fragment thereof) whichbinds specifically to C. immitis but not H. capsulatum, B. dermatitidis,P. brasiliensis, or P. marneffei; the Pb probe (or a variant or fragmentthereof) which binds specifically to H. capsulatum, B. dermatitidis, C.immitis, or P. marneffei; and/or the Pm probe (or a variant or fragmentthereof) which binds specifically to P. marneffei but not H. capsulatum,B. dermatitidis, C. immitis, or P. brasiliensis.

As used herein, each species-specific or microbe-specific probe refersto a probe that binds to the nucleic acid sequence of a species with aspecificity sufficient to distinguish different species from oneanother. In particular examples, that specificity is at least 3.0, or atleast 7.0, as measured by an EIA index (EI) equal to the optical densityof the test DNA (i.e., the probe tested) divided by the optical densityof a water blank. One particular, non-limiting example of determining EIis provided in Example 10.

In certain examples, a probe can bind to two species detectably (as withBd which binds to B. dermatitidis with a higher specificity (11.9) thanit binds to C. immitis (4.3)), but the higher specificity can be used todistinguish the two. Alternatively, a specimen with nucleic acid thatdetectably binds to the Bd probe also can be probed with the Ci probe,to distinguish B. dermatitidis from C. immitis infection.

When referring to species-specific or microbe-specific probes, it isunderstood that this refers to a probe having a specificity of probebinding of at least 3.0 EI for the nucleic acid of the fungus ofinterest, such as H. capsulatum, B. dermatitidis, C immitis, P.brasiliensis, or P. marneffei. Particular, non-limiting, examples of themicrobe-specific probes are Hc, Ci, or Pb, while Pm is a particular,non-limiting, example of a species-specific probe. However,species-specific and microbe-specific probes also refer to variants,fragments, and longer probes that contain the species-specific ormicrobe-specific sequences of the disclosed sequences.

A probe specific for a dimorphic fungus refers to a probe having asequence that binds to the nucleic acid of a dimorphic fungus withsufficient specificity to distinguish the fungus from a non-dimorphicfungus, such as S. schenckii, C. neoformans, or other organism, such asP. carinii. In particular examples, the probe specifically binds to thedimorphic fungus with a specificity of at least 2.5, 3.0, or 5.0 EI.

A “variant” of a probe includes sequences that have altered nucleic acidsequences, but retain their ability to bind to the target sequences (andidentify the fungal target) with sufficient specificity. In someparticular examples, no more than 1, 2, 5, or 10 nucleic acids arechanged, or the probe retains at least 80%, 85%, 90%, or 95% sequenceidentity to the original probe. Variants also include probe sequences towhich an additional nucleic acid sequence has been added, while stillretaining the noted specificity of the probes. Fragments includeshortened probe sequences (or subsequences) of a probe that also retainsthe noted specificity.

Any of these variants or fragments can be screened for retention ofspecificity by determining the EI of the variant or fragment, such as(and without limitation) by the assay described in Example 10. However,because the EI is based on a ratio, and not an absolute measurement,other techniques for measuring hybridization, rather than opticaldensity of a colored dye, can be used.

Kits

The oligonucleotide primers and probes disclosed herein can be suppliedin the form of a kit for use in detection of fungi, including kits forany of the arrays described above. In such a kit, an appropriate amountof one or more of the oligonucleotide primers and/or probes is providedin one or more containers or held on a substrate. An oligonucleotideprimer or probe can be provided suspended in an aqueous solution or as afreeze-dried or lyophilized powder, for instance. The container(s) inwhich the oligonucleotide(s) are supplied can be any conventionalcontainer that is capable of holding the supplied form, for instance,microfuge tubes, ampules, or bottles. In some applications, pairs ofprimers can be provided in pre-measured single use amounts inindividual, typically disposable, tubes or equivalent containers. Withsuch an arrangement, the sample to be tested for the presence of fungalnucleic acids can be added to the individual tubes and amplificationcarried out directly.

The amount of each oligonucleotide primer supplied in the kit can be anyappropriate amount, and can depend on the target market to which theproduct is directed. For instance, if the kit is adapted for research orclinical use, the amount of each oligonucleotide primer provided wouldlikely be an amount sufficient to prime several PCR amplificationreactions. General guidelines for determining appropriate amounts can befound in Innis et al., Sambrook et al., and Ausubel et al. A kit caninclude more than two primers in order to facilitate the PCRamplification of a larger number of fungal nucleotide sequences.

In some embodiments, kits also can include the reagents necessary tocarry out PCR amplification reactions, including DNA sample preparationreagents, appropriate buffers (e.g., polymerase buffer), salts (e.g.,magnesium chloride), and deoxyribonucleotides (dNTPs).

Kits can include either labeled or unlabeled oligonucleotide probes foruse in detection of fungal nucleotide sequences. The appropriatesequences for such a probe will be any sequence that falls between theannealing sites of the two provided oligonucleotide primers, such thatthe sequence that the probe is complementary to is amplified during thePCR reaction. In some embodiments, the probe is complementary to asequence within the fungal ITS2 region.

One or more control sequences for use in the PCR reactions also can besupplied in the kit. Appropriate positive control sequences can beessentially as those discussed above.

Particular embodiments include a kit for detecting and identifying afungus based on the arrays described above. Such a kit includes at leasttwo different probes (as described above) and instructions. A kit cancontain more than two different probes, such as at least 10, at least25, at least 50, at least 100, or more probes. The instructions caninclude directions for obtaining a sample, processing the sample,preparing the probes, and/or contacting each probe with an aliquot ofthe sample. In certain embodiments, the kit includes a device orapparatus for separating the different probes, such as individualcontainers (e.g., microtubules) or an array substrate (e.g., a 96-wellor 384-well microtiter plate). In particular embodiments, the kitincludes prepackaged probes, such as probes suspended in suitable mediumin individual containers (e.g., individually sealed Eppendorf® tubes) orthe wells of an array substrate (e.g., a 96-well microtiter plate sealedwith a protective plastic film). In other particular embodiments, thekit includes equipment, reagents, and instructions for extracting and/orpurifying nucleotides from a sample.

EXAMPLES

The following examples are provided to illustrate particular features ofcertain embodiments, but the scope of the claims should not be limitedto those features exemplified.

Example 1 Isolation of Fungal DNA

One loopful of yeast phase B. dermatitidis (strains 4478, KL-1 (ATCC26198), or A2 (ATCC 60916)), was inoculated into 10 ml of Brain HeartInfusion broth (Difco, Becton Dickinson, Sparks, Md.) in a 50 μlErlenmeyer flask and incubated at 37° C. on a rotary shaker (140 rpm)for 48 to 72 h. The suspension was then transferred to a 30 mlcentrifuge tube (Oak Ridge, Nalge, Rochester, N.Y.) and centrifuged for3 min at 2000×g. Genomic DNA was extracted and purified using acommercial kit (PureGene Yeast and Gram Positive DNA Isolation Kit;Gentra Systems Inc., Minneapolis, Minn.) following the manufacturer'sprotocol.

Mold phase cultures of Sporothrix schenckii (ATCC 58251) and Penicilliummarneffei (strains ATCC 64101, ATCC 58950, and JH05 (gift of Dr. WilliamMerz, Johns Hopkins Medical School, Baltimore, Md.)) were grown in 50 mlof Sabouraud dextrose broth (Difco) in 250 ml Erlenmeyer flasks andincubated at 25° C. on a rotary shaker for 5 days. Growth was harvestedby vacuum filtration through sterile filter paper, and the cellular matwas washed three times with sterile distilled H₂O by filtration. Thecellular mat was then removed from the filter and placed into a sterilePetri plate, which was then sealed around the edges with Parafilm®(American Can, Neenah, Wis.) and frozen at −20° C. until used.

DNA was extracted by grinding the cellular mats with a mortar and pestlein the presence of liquid nitrogen. Just before use, a portion of thefrozen cellular mat, approximately an inch in diameter, was removed fromthe Petri plate with sterile forceps and placed into an ice-cold,sterile mortar (6 inches diameter). Liquid nitrogen was added to coverthe mat and was added as needed to keep the mat frozen during grinding.The fungal mat was ground into a fine powder with a sterile pestle.Fungal DNA was then extracted and purified using serial proteinase K andRNase treatments followed by phenol extraction and ethanol precipitationby standard methods. See Maniatis, et al., Molecular Cloning: ALaboratory Manual (Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1982).

Other DNA was kindly provided as a gift from the following persons:Histoplasma capsulatum (strains G186B (ATCC26030), Downs, FLs-1, andB295 (var. duboisii)), Dr. Brent Lasker, Centers for Disease Control andPrevention (CDC), Atlanta, Ga.; Coccidioides immitis (strains C635 andC735), Dr. Garry Cole, Medical College of Ohio, Toledo, Ohio;Paracoccidioides brasiliensis (strains 265, Pb18, rh, soil (soil isolatefrom Venezuela)), Dr. Maria Jose Soares Mendes Giannini, Faculdade deCiencias Farmaceuticas, UNESP, Araraquara, Brazil and Dr. Juan McEwen;Corporacion Para Investigaciones Biologicas, Medellin, Colombia; C.neoformans (strains 9759-MU-1 (serotype A), BIH409 (serotype B),K24066TAN (serotype C) and 9375 (serotype D)) and all Candida speciesDNA (Candida albicans (strain B311), Candida glabrata (CDC Y-65) Candidakrusei (CDC 259-75), Candida tropicalis (CDC 38), and Candidaparapsilosis (ATCC 22019)), Ms. Cheryl Elie, CDC; and Pneumocystiscarinii (rat isolate), Dr. Charles Beard, CDC.

Example 2 High-Throughput Preparation of Fungal DNA

Fungi

Aspergillus fumigatus (ATCC 42202); Candida albicans (CBS-2730);Fusarium solani (ATCC 52628); Mucor racemosus (ATCC 22365);Pseudallescheria boydii (ATCC 36282); Sporothrix schenckii (ATCC 28184).Candida albicans was cultured on Sabouraud dextrose agar at 37° C.,Sporothrix schenckii was cultured on potato dextrose agar at 27° C., andthe remaining four species were cultured on potato dextrose agar at 37°C.

High-Throughput DNA Extraction

Three types of acid-washed glass beads were added to each well of asterile 96 deep-well plate capable of holding 2 ml of sample per well(Bellco Glass, Inc., Vineland, N.J.). The amounts and types of glassbeads used were: (1) 100 μl of 106 μm glass beads (Sigma Chemical Co.,St Louis, Mo.); (2) 100 μl of 3 mm glass beads (Corning, Inc., Corning,N.Y.); and (3) 150 μl of 0.5 mm glass beads (BioSpec Products, Inc.,Bartlesville, Okla.). Cells or conidia (10⁸) of the fungi listed above,suspended in 400 μl of TSTE buffer, were then added to each well. Theplate was sealed with a cap mat and shaken for 30 min at 500 rpm in arotary incubator (New Brunswick Scientific, Edison, N.J.) fitted with astainless steel utility tray to hold the plate.

DNA Purification

Extracted DNA from each well was purified by first diluting the solutionfrom the well in 300 μl of 6 M NaCl in a 2 ml centrifuge tube andvortexing for 30 seconds. Each tube was centrifuged at 2750×g for 30minutes, and about 700 μl of the supernatant was transferred to a fresh1.5 ml Eppendorf® tube where it was washed by centrifugation with 700 μlof isopropanol. The supernatant was removed and 300 μl of 70% ethanolwas added to the tube. The sample was again centrifuged at 10,000×g for30 minutes, the supernatant removed, and the pellet was air dried for 30min. After air-drying, 50 μl TE and 1 μl of 500 μg/ml of RNase wasadded. The sample was then incubated at 37° C. for 30 min. The DNA wasthen resuspended in 50 μl TE buffer.

DNA Quantitation

The quantity of DNA isolated in each sample was estimated using theA_(260/280nm) ratio. Absorbance measurements were accomplished using aHoefer DyNA™ 200 fluorometer with 2 μl of Hoescht 333258 dye diluted forlow-range DNA detection (Amersham Pharmacia Biotech, Inc., Piscataway,N.J.) added to each 2 μl of sample.

PCR Amplification

The ITS3 and ITS4 universal fungal primers were used to amplify a regionof the fungal rRNA gene (see FIG. 1). The reaction mixture contained 5μl of 10× PCR buffer; 1 μl of dNTPs (0.2 μM); 0.5 μl of each primer (20μM); 2.5 U of Taq DNA polymerase (Roche); and 2 μl of template DNA (5ng). Samples were placed in a Perkin-Elmer 9600 thermal cycler at 95° C.for 30 minutes to denature DNA and then for 36 cycles of 95° C. for 30seconds (denaturation); 58° C. for 30 seconds (annealing); 72° C. for 1minute (extension); and a final extension step of 72° C. for 10 minuteswere conducted.

Example 3 Comparison of High-Throughput Preparation of Fungal DNA withStandard Methods

The high-throughput technique described in Example 2 was compared withother standard DNA extraction techniques.

DNA Extraction by Enzymatic Digestion

Enzymatic digestion with lyticase was performed by gentle vortex mixingafter suspending 10⁸ cells or conidia in 500 μl of lysis buffer A (IMsorbitol, 0.05 M sodium phosphate (monobasic), 0.1% 2-mercaptoethanol,100 μg/ml lyticase (Sigma Chemical Co.)). Cell suspensions were thenincubated in a stationary mode for 3 h at 37° C. Nuclei were lysed bythe addition of 100 μl of lysis buffer B (10% SDS and 0.05 M EDTA (pH8.0) (Sigma Chemical Co.)).

DNA Extraction by Glass Bead Beater

Cells or conidia (10⁸) were suspended in 400 μl of TSTE buffer (5%Triton X-100, 100 mM NaCl, 10 mM Tris (pH 8.0), 1 mM EDTA) andtransferred to a 2 ml tube containing 400 μl of glass beads (Q-BIOgene,Carlsbad, Calif.). TSTE buffer (400 μl; see above) was added and thetube was placed in a FastPrep® instrument (Q-BIOgene). The sample wasprocessed for 45 seconds on the highest setting and then cooled on icefor 5 minutes.

DNA Extraction by Liquid Nitrogen Grinding

This method was carried out under a fume hood. Cells or conidia (10⁸)were transferred into a pre-cooled, sterile mortar using a sterileinoculating loop. Five ml of liquid nitrogen was added, cells werefrozen and manually ground into a fine powder using a sterile pestle.One ml of TSTE buffer (see above) was added, and the mortar and pestlewere allowed to soak in the buffer for 30 minutes before transfer of theliquid to a 2 ml centrifuge tube.

Comparison Results

DNA was purified and amplified by PCR substantially as described inExample 2, except that the initial centrifugation step of DNApurification was carried out at 10,000×g for 30 minutes, rather than2750×g for 30 minutes as used in the high-throughput method.

DNA extraction by the high-throughput method described in Example 2 wasunexpectedly superior to the other three methods—enzymatic digestion,glass bead beater, and liquid nitrogen grinding. The high-throughputmethod required minimal production time (less than 2 hours, compared toup to 4 hours for the other DNA extraction methods), needed nospecialized equipment (unlike the glass bead beater method, whichrequires a FastPrep instrument), and up to 96 samples could be processedat once (compared to only a single sample or a few samples for theenzymatic digestion and liquid nitrogen grinding method, and up to 12samples for the glass bead beater method). Furthermore, thehigh-throughput method produced a DNA yield suitable for typing purposes(30±2 ng/μl, n=6) and no expensive enzymes were needed (compared to theenzymatic degradation method). FIG. 4 is a digital print of an agarosegel showing PCR products after DNA isolation using the high-throughputmethod. Lane M is a 100-bp DNA ladder (1000 to 100 bp), while lanes 1 to6 are amplicons from F. solani, M. racemosus, S. schenckii, A.fumigatus, P. boydii, and C. albicans, respectively. FIG. 5 is a digitalprint of an agarose gel demonstrating PCR sensitivity based on (A) A.fumigatus (B) C. albicans genomic DNA isolated by the high-throughputmethod. Lanes 1 to 6, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, and 1 fg ofDNA, respectively, after PCR amplification. Lane 7, water control. LaneM, 100-bp DNA ladder (1000 to 100 bp).

Example 4 Preparation of Primers and Probes

All primers and probes were synthesized by β-cyanoethyl phosphoramiditechemistry using a 394 or expedite automated DNA synthesizer (PE AppliedBiosystems, Foster City, Calif.). ITS3, a universal fungal sequencelocated in the 5.8S region of the rRNA gene and contained within theregion amplified by ITS1 and ITS4 primers (see Lott, et al., Yeast9:1199-206 (1993); and White, T. J., et al., in M. A. Innis, et al.(ed.), PCR Protocols: A guide to methods and applications (AcademicPress, San Diego, Calif., 1990) was biotinylated at the 5′ end byincorporating dimethyoxytrityl-biotin-carbon-6-phosphoramidite duringits synthesis. This biotinylated probe (ITS3-B) was then purified byreverse phase liquid chromatography. Digoxigenin-labeled probes weresynthesized with a 5′-terminal amine group using 5′ Amino-Modifier C6(Glen Research, Sterling, Va.), mixed with a 10-fold molar excess ofdigoxigenin-3-O-methylcarbonyl-ε-aminocaproic acid N-hydroxysuccinimideester (Roche Molecular Biochemicals, Indianapolis, Ind.) in 0.1 M sodiumcarbonate buffer, pH 9.0, and incubated at ambient temperatureovernight. The digoxigenin-labeled probes were then purified byreverse-phase high pressure liquid chromatography. See Becker, et al.,J. Chromatogr. 326:293-299 (1985).

Sequences and locations in the rRNA gene of these primers and probes aredepicted in Table 1 and FIG. 1, respectively. All primers and probeswere synthesized by the CDC Biotechnology Core Facility.

Example 5 Microbe-Specific Probes

DNA sequences of the ITS2 region of the fungal rRNA gene (see FIG. 1)were obtained from GenBank and are listed in Table 1. TABLE 1 Sequencesof oligonucleotide primers and probes. SEQ SEQUENCE (5′ to 3′) ID NO:OLIGONUCLEOTIDE LABELING PCR PRIMERS ITS1 TCCGTAGGTGAACCTGCGG 1Universal forward primer ITS4 TCCTCCGCTTATTGATATGC 2 Universal reverseprimer PROBES ITS3-B GCATCGATGAAGAACGCAGC 3 5′-biotin-labeled universalcapture probe Dm GGACGTGCCCGAAATGCAGTGGCGG 4 5′-digoxigenin-labeledprobe for all endemic dimorphic fungi Hc ACCATCTCAACCTCCTTTTTCACACCAGG 55′-digoxigenin-labeled probe for Histoplasma capsulatum BdGGTCTTCGGGCCGGTCTCCCC 6 5′-digoxigenin-labeled probe for Blastomycesdermatitidis Ci CTCTTTTTTTTATTATATCC 7 5′-digoxigenin-labeled probe forCoccidioides immitis Pb CACTCATGGACCCCGG 8 5′-digoxigenin-labeled probefor Paracoccidioides brasiliensis Ss GACGCGCAGCTCTTTTTA 95′-digoxigenin-labeled probe for Sporothrix schenckii PmGGGTTGGTCACCACCATA 10  5′-digoxigenin-labeled probe for Penicilliummarneffei Cn CCTATGGGGTAGTCTTCGG 11  5′-digoxigenin-labeled probe forCryptococcus neoformans Pc GTAGTAGGGTTAATTCAATT 12 5′-digoxigenin-labeled probe for Pneumocystis carinii

Those fungi that did not have sequences available in GenBank (P.brasiliensis, S. schenckii and P. marneffei) were sequenced using a dyeterminator cycle sequencing kit (ABI PRISM, Applied Biosystems, PerkinElmer, Foster City, Calif.) and sequences have since been deposited withGenBank by our laboratory or by others (Accession numbers: Sporothrixschenckii, AF117945; P. brasilieisis, AF322389; P. marneffei, L37406).Briefly, primary DNA amplifications were conducted using ITS1 and ITS4as primers. The DNA was purified using QIAquick Spin Columns (QiagenCorp., Chatsworth, Calif.) and eluted with 50 ml of heat-sterilizedTris-EDTA buffer (10 mM Tris, 1 mM EDTA, pH 8.0). Sequencing wasperformed in both the forward and the reverse directions. The PCRreaction mix (20 μl) containing 9.5 μl terminator premix, 2 μl (1 ng)DNA template, 1 μl primer (either a forward or reverse primer, 3.2pmol), and 7.5 μl heat-sterilized distilled H₂O was placed into apre-heated (96° C.) Perkin-Elmer 9600 thermal cycler for 25 cycles of96° C. for 10 sec, 50° C. for 5 sec, and 60° C. for 4 min. The PCRproduct was then purified before sequencing using CentriSep spin columns(Princeton Separations, Inc., Adelphia, N.J.). DNA was then vacuumdried, resuspended in 6 μl of formamide-EDTA (5 μl deionized formamideplus 1 μl of 50 mM EDTA, pH 8.0), and denatured for 2 min at 90° C.before subjection to sequencing using an automated capillary DNAsequencer (ABI Systems, Model 373, Bethesda, Md.).

Sequences were aligned, and a comparison was performed to determineunique sequences that could be used for the development of specificdigoxigenin-labeled oligonucleotide probes. The initial screen forspecificity of the probe sequences was performed using BLAST software(GCG, Madison, Wis.). Probe sequences determined to be unique were thensynthesized and digoxigenin-labeled as described above.

Example 6 PCR Amplification using ITS1 and ITS4 as Primers

The PCR reaction mix consisted of 10 mM Tris-HCl buffer containing 50 mMKCl, pH 8.0 (Roche), 1.5 mM MgCl₂ (Roche), 0.2 mM dNTP (TaKaRa Shuzo Co.Ltd; Otsu, Shiga, Japan) and 1.25 U Taq polymerase (TaKaRa Shuzo).Primers ITS1 and ITS4 were added to a final concentration of 0.2 mMeach. Template DNA was added at a final concentration of 1 ng per 50 μlreaction mix. For each experiment, at least one reaction tube receivedwater in place of template DNA as a negative control. Amplification wasperformed in a Model 9600 thermocycler (Perkin Elmer, EmeryvilleCalif.). Initial denaturation of template DNA was achieved by heating at95° C. for 5 minutes. This was followed by 30 cycles of 30 s at 95° C.,30 s at 58° C., and 1 min at 72° C. A final extension step was conductedfor 10 min at 72° C. Appropriate controls were included and PCRcontamination precautions were followed. Fujita, S., et al., J. Clin.Microbiol. 33:962-67 (1995); Kwok, S., and R. Higuchi, Nature 339:237-38(1989).

Example 7 Confirming PCR Amplification by Agarose Gel Electrophoresis

To verify that the specific DNA target was appropriately amplified andwas of the expected size, the PCR amplicons were subjected to agarosegel electrophoresis and bands were visualized after ethidium bromidestaining.

Gels consisted of 1% agarose LE (Boehringer Mannheim, Indianapolis,Ind.) and 1% NuSieve GTG agarose (FMC Bioproducts, Rockland, Me.) or 2%Metaphore agar (FMC Bioproducts) dissolved in TBE buffer (0.1 M Tris,0.09 M boric acid, 0.001 M EDTA, pH 8.4). Five microliters of the PCRamplicons were combined with 1 μl of tracking dye (Roche) and then addedto each well of the agarose gel. Electrophoresis was conducted at 70-80V for 45-60 minutes. The gel was stained with ethidium bromide for 30min and washed in deionized water for 30 min before examination on a UVtransilluminator.

The amplification of the rRNA gene using the ITS1 and ITS4 primersresulted in an approximately 600 bp amplicon for all fungi tested. Asillustrated in FIG. 7, the molecular sizes of amplicons were especiallysimilar among the dimorphic fungi. In FIG. 7, lane abbreviations are(from left to right): MW, molecular wt markers (HaeIII digest of φX174plasmid, Roche, Indianapolis, Ind.); H. capsulatum, DNA amplified fromH. capsulatum strains B293 (var. duboisii), Down's, and Fls-1; B.dermatitidis, DNA amplified from B. dermatitidis strains 4478, KL1, andA2; C. immitis, DNA amplified from C. immitis strains C635 and C735; C.neoformans, DNA amplified from C. neoformans strains 9759-MU-1 (serotypeA), BIH409 (serotype B), K24066TAN (serotype C), and 9375 (serotype D);lanes CA, CG, CK, CT, and CP, DNA amplified from C. albicans (strainB311), C. glabrata (CDC Y-65), C. krusei (CDC 259-75), C. tropicalis(strain CDC 38), and C. parapsilosis (ATCC 22019), respectively; lane B,water blank (negative control). The greatest differences in ampliconsize were observed among the five Candida species tested and wereparticularly pronounced for C. glabrata and C. krusei compared to allother Candida species. However, specific identification of the fungiusing amplicon size alone was not possible and is not generallyrecommended. Podzorski, R. P., and D. H. Persing, In P. R. Murray, etal. (eds.), Manual of Clinical Microbiology, 6th ed. (ASM Press,Washington, D.C., 1995), 130-157. Therefore, probes were designed tospecifically identify each fungus using the PCR-EIA identificationmethod described in Example 8.

Example 8 Polymerase Chain Reaction-Enzyme Immunoassay (PCR-EIA)

EIA identification of PCR products was performed as described in Elie,C. M., et al., J. Clin. Microbiol. 36:3260-65 (1998) and Fujita, S., etal., J. Clin. Microbiol. 33:962-7 (1995), with minor modifications.FIGS. 1A-B illustrate this protocol. Briefly, tubes containing 10 μl ofheat-denatured (5 min at 95° C.) PCR amplicons were placed on ice, and200 μl of hybridization buffer (4×SSC, pH 7.0, 0.02M HEPES, 0.002M EDTA,0.15% Tween 20) containing 10 ng of ITS3-B and 10 ng of adigoxigenin-labeled specific probe was added. Samples were mixed andincubated at 37° C. for 1 h. One hundred microliters of the mixture wasadded in duplicate to each well of a streptavidin-coated, 96-well,microtiter plate (Roche) and incubated at ambient temperature for 1 h ona microtiter plate shaker (˜350 rpm, Labline Instruments, Melrose Park,Ill.). Microtiter plates were washed 6 times with 0.01 M phosphatebuffered saline, pH 7.2 (GibcoBRL, Life Technologies, Grand Island,N.Y.), containing 0.05% Tween 20 (Sigma Chemical Co., St Louis, Mo.)(PBST) before adding 100 μl of a 1:1000 dilution of horseradishperoxidase-labeled, anti-digoxigenin antibody (150 U/ml, Roche) perwell. Plates were incubated for 1 h at ambient temperature with shakingand then washed 6 times with PBST. 3,3′,5,5′-Tetramethylbenzidine(TMB)-H₂O₂ substrate (Kirkegaard and Perry, Gaithersburg, Md.) was thenadded to the wells and the color reaction was allowed to develop atambient temperature for 15 min. The optical density of each well wasimmediately read at a wavelength of 650 nm in a UVMax microtiter platereader (Molecular Devices, Sunnyvale, Calif.). The optical density ofthe duplicate wells were averaged and used in the analysis of theresults. The optical density results were then converted to an EIA index(EI) which was calculated by dividing the optical density value of thewells which had received test DNA by the optical density of the PCRwater control: O.D. of test DNA/O.D. of water blank=EI.

Example 9 Statistical Analysis

Student's t test was used to determine differences between the mean EIof probe hybridization to homologous and heterologous DNA. Differenceswere considered significant when the value of P was less than or equalto 0.05.

Example 10 Probe Specificity

Digoxigenin-labeled probes directed to the ITS2 region of rDNA weredesigned to specifically detect PCR amplicons from the most medicallyimportant yeast-like fungi. In addition to the microbe-specific probes,a probe was also designed as a primary screening probe (Dm; SEQ ID NO:4) with which to identify only the systemic, dimorphic fungal pathogens.The specificity of these probes was confirmed using the PCR-EIA methodin a checkerboard pattern as shown in Table 2. TABLE 2 Specificity ofoligonucleotide probes to DNA from yeast-like fungi Mean EI ± S.E. (n)using DNA from: H. B. C. P. P. S. C. P. Candida PROBE capsulatumdermatitidis immitis brasiliensis marneffei schenckii neoformans ^(b)carinii species^(c) Dm 11.0 ± 1.1  9.4 ± 1.4 16.9 ± 2.2 13.9 ± 1.1 3.0 ±0.5  0^(d) 0 0 0 (34) (24) (14) (21) (31) Hc 15.8 ± 1.4 0 0 0 0 0 0 0 0(37) Bd 0 11.9 ± 2.0  4.3 ± 0.8 0 0 0 0 0 0 (20) (10) Ci 0 0 21.9 ± 3.20 0 0 0 0 0 (13) Pb 0 0 0 10.8 ± 0.8 0 0 0 0 0 (22) Pm 0 0 0 0 7.6 ± 0.60 0 0 0 (36) Ss 0 0 0 0 0 23.6 ± 4.3 0 0 0 (12) Cn 0 0 0 0 0 0 42.7 ±2.7 0 0 (31) Pc 0 0 0 0 0 0 0 6.2 ± 2.7 0 (10)^(a)See Table 1 for definition of abbreviations.^(b)Includes DNA from serotypes A, B, C, and D.^(c) Candida species included: C. albicans, C. glabrata, C. krusei, C.tropicalis, and C. parapsilosis; excludes results for Hc probe# against C. albicans: mean EI ± S.E. = 6.5 ± 1.0, (n = 10).^(d)A value of zero was assigned for all EI values less than 1.75 forease of presentation. Mean EIA ± S.E. (n) for all heterologous DNAtested with the following# probes: Dm, 1.13 ± 0.04 (n = 89); Hc, 1.2 ± 0.3 (n = 118); Bd, 1.41 ±0.12 (n = 103); Ci, 1.0 ± 0.01 (n = 96); Pb, 1.01 ± 0.02 # (n = 88); Pm,0.99 ± 0.02 (n = 98); Ss, 0.99 ± 0.01 (n = 103); Cn, 0.98 ± 0.01 (n =95); and Pc, 0.98 ± 0.01 (n = 96). All probes # significantly hybridizedto homologous DNA but not heterologous DNA at P < 0.001 except for Pc (P< 0.05) and Bd vs. C. immitis DNA (P < 0.01).

The dimorphic screening probe (Dm) successfully hybridized with PCRamplicons from all strains of the major systemic, dimorphic fungi tested(H. capsulatum, B. dermatitidis, C. immitis, P. brasiliensis, and P.marneffei) but not with DNA from any strain of the other yeast-likefungi (S. schenckii, C. neoformans, Canidida species or P. carinii).

Microbe-specific probes (Hc, Bd, Ci, Pb, Pm, Ss, Cn, and Pc), designedto detect only DNA amplified from their homologous fungus, were testedagainst PCR amplicons from all strains of both homologous as well asheterologous yeast-like fungi. The results in Table 2 demonstrate thatthe microbe-specific probes hybridized with DNA from homologous fungiand not with DNA from heterologous fungi (P<0.001 or P<0.05) with minorexceptions. There was some reactivity of the B. dermatitidis probeobserved when it was tested against C. immitis DNA. However, thehybridization signal for the B. dermatitidis probe tested against B.dermatitidis DNA was statistically greater than that for C. immitis DNA(11.9±2.0 versus 4.3±0.8, P <0.01). In addition, the reverse (i.e., theC. immitis probe tested against B. dermatitidis DNA) was negative andcould be used to differentiate the two fungi by a process ofelimination. There was also a hybridization signal observed for the H.capsulatum probe reacted with DNA from C. albicans (15.8±1.4 versus6.5±1.0, P<0.001), but no signal was observed for any of the otherCandida species tested using this probe. The dimorphic probe, however,did not hybridize with C. albicans DNA and the C. albicans probe doesnot hybridize with H. capsulatum DNA (data not shown; see Elie, et al.,and Fujita, et al.).

Example 11 Confirmation of Probe Specificity using Multiple Strains ofHomologous and Heterologous Fungi

To further analyze each probe's capacity to hybridize with only DNA fromhomologous fungi, DNA from multiple strains of each of the systemicdimorphic fungi were tested in the PCR-EIA. Results are shown in Table3. TABLE 3 Reactivity of oligonucleotide probes to dimorphic pathogensagainst DNA from multiple strains of homologous and heterologousdimorphic fungi. Mean EI ± S.E.(n) using DNA from^(a): H. capsulatumisolates B. dermatitidis isolates PROBE^(b) 1 2 3 4 1 2 3 Dm 13.7 ± 2.8 8.8 ± 1.8 10.3 ± 2.0 11.4 ± 2.8 9.2 ± 1.8 12.5 ± 3.0 4.9 ± 0.8 (8) (7)(11) (8) (9) (9) (6) Hc 17.3 ± 3.8 15.1 ± 2.3 15.8 ± 2.6 14.7 ± 2.6 0^(c) 0 0 (9) (6) (10) (9) Bd 0 0 0 0 9.7 ± 2.4 15.4 ± 3.5 8.3 ± 3.3(6) (9) (5) Ci 0 0 0 0 0 0 0 Pb 0 0 0 0 0 0 0 Mean EI ± S.E.(n) usingDNA from^(a): C. immitis isolates P. brasiliensis isolates PROBE^(b) 1 21 2 3 Dm 14.8 ± 1.8 18.5 ± 3.6 13.3 ± 2.2 14.1 ± 2.3 14.3 ± 2.0 (6) (8)(7) (7) (6) Hc 0 0 0 0 0 Bd  4.7 ± 1.2  3.8 ± 1.2 0 0 0 (5) (5) Ci 23.8± 4.2 20.2 ± 4.9 0 0 0 (6) (7) Pb 0 0  9.4 ± 1.5 12.3 ± 1.4 12.0 ± 0.4(7) (7) (6)^(a)Isolates used: H. capsulatum: 1) G186B, 2) B293, 3) Downs, 4)FLs1;B. dermatitidis: 1) 4478, 2) A2, 3) KL-1; C. immitis: 1) C634, 2) C735;P. brasiliensis: 1) Pb18, 2) rh, 3) soil.^(b)See Table 1 for definition of abbreviations.^(c)A value of zero was assigned for all EI values less than 1.75 forease of presentation. Mean EI ± S.E. (n) for heterologous DNA testedwith the following# probes: Hc, 1.2 ± 0.05 (n = 42); Bd, 1.74 ± 0.27 (n = 44); Bd withoutCi DNA 0.99 ± 0.02 (n = 34); Ci, 1.0 ± 0.02 (n = 42); and Pb, 0.99 ±0.03 (n = 33). All # probes significantly hybridized to homologous DNAbut not heterologous endemic dimorphic DNA at P<0.001

The probe designed to identify all systemic dimorphic fungi (Dm)hybridized with DNA from all strains of H. capsulatum, B. dermatitidis,C. immitis, and P. brasiliensis tested. In addition, the probes specificfor individual dimorphic fungi (Hc, Bd, Ci, Pb) hybridized only to DNAisolated from homologous fungi, but not DNA isolated from heterologousfungi. The minor hybridization signal observed for the B. dermatitidisprobe tested against C. immitis DNA was similar for both strains of C.immitis tested.

Example 12 Sensitivity of Probes using the PCR-EIA Method

To assess the limit of sensitivity of the PCR-EIA method, compared tothat for detection of amplicons by agarose gel electrophoresis, H.capsulatum (Down's strain) DNA was serially diluted prior to PCRamplification and then assessed by both agarose gel electrophoresis andPCR-EIA. FIG. 6 is a digital print of an agarose gel, stained withethidium bromide, demonstrating detection of amplicons at aconcentration as low as 16 pg of DNA. In FIG. 6, lane 1 containsmolecular size markers (AmpliSize molecular ruler, BioRad, Hercules,Calif.); lanes 2 to 8, pg of DNA per reaction: 20,000; 10,000; 2,000;400; 80; 16; and 3.2, respectively. In contrast, as little as 3.2 pg ofDNA could be detected by PCR-EIA as demonstrated in Table 4. TABLE 4Evaluation of the sensitivity of the PCR-EIA assay. DNA conc^(a) EIAIndex^(b) Agarose gel^(c) 10,000 53.3 + 2,000 50.4 + 400 38.2 + 8013.9 + 16 5.3 + 3.2 2.2 − 0.64 1.53 − 0.128 1.15 − 0.0256 1.06 −^(a)pg/reaction^(b)EIA index using H. capsulatum (Downs strain) DNA^(c)+ = visually positive band in gel after EtBr staining

Example 13 Differentiation of Penicillium marneffei

Microorganisms

Clinical isolates, or cultures obtained from the American Type CultureCollection (ATCC), or the CDC Mycotic Diseases Branch Laboratory, wereused. See Table 5 below.

DNA Isolation

DNA was extracted from all species by using the FastDNA kit (Q-BIOgene,Carlsbad, Calif.) with minor modification.

Design of Probes

Universal fungal primers, ITS3 and ITS4, were used to amplify the ITS2rDNA region. Oligonucleotide probes were designed from GenBank sequencedata for the ITS2 region of Penicillium species, or were sequenced bystandard capillary methods if sequences were not available in GenBank.

PCR Amplification

The reaction mixture contained 5 μl of 10× PCR buffer, 0.2 μM dNTPs, 0.5μl of each primer (20 μM), Taq DNA polymerase (2.5 U, Roche), templateDNA (5 ng), and sterile distilled water to bring the volume to 50 μl.PCR amplification conditions were 5 min of denaturation at 95° C.,followed by 30 cycles of 95° C. for 30 s, 58° C. for 30 s, and 72° C.for 1 min, carried out on a Perkin-Elmer thermal cycler described above.A final extension step of 72° C. for 5 min was then conducted.Electrophoresis was carried out at 80 V for approximately 1 h in gelscomposed of 1% (wt/vol) agarose. Gels were stained with ethidiumbromide, visualized with a UV transilluminator, and photographed.

EIA

PCR-amplified DNA was hybridized to species-specific digoxigenin-labeledprobes and a universal biotinylated probe, and then the complex wasadded to streptavidin-coated microtitration plates and captured. Acolorimetric EIA was then conducted to detect captured DNA by usinghorseradish peroxidase-conjugated anti-digoxigenin antibody and TMB-H₂O₂substrate, as illustrated in FIGS. 1A-B.

Statistical Analyses

Student's t test was used to determine significant differences betweenmean absorbance values of homologous and heterologous DNA reactions withprobes. Differences were considered significant when the value of P wasless than or equal to 0.05. TABLE 5 Source and characteristics ofisolates. Identification Isolate Number Source/CharacteristicsPenicillium spp. P. marneffei ATCC 18224 Type culture, isolated frombamboo rat P. marneffei B6006 CDC, CSF from AIDS patient P. marneffeiB6015 CDC, human skin biopsy P. marneffei JH05 Gift from W. Merz, humanskin lesion P. marneffei B6010 CDC P. camembertii ATCC 4845 Camembertcheese, France P. caseicolum ATCC 6986 Camembert cheese P. chrysogenumATCC 10106 Produces chrysogenin P. citrinum B5809 CDC P. glabrum ATCC16349 Production of acid protease P. griseofulvum ATCC 66967Penicilliosis patient P. italicum ATCC 48114 From fruit P. janthinellumATCC 10069 Soil P. purpurescens ATCC 20075 Soil P. purpurogenum ATCC10064 Unknown P. roquefortii ATCC 10110 Roquefort cheese, France P.rubefaciens ATCC 48481 Soil P. spinulosum ATCC 16348 Tanning liquorAspergillus spp. A. flavus ATCC 64025 Human sputum A. fumigatus ATCC42202 Human sputum A. nidulans ATCC 10074 Unknown A. niger ATCC 16888Tannin-gallic acid fermentation A. terreus ATCC 1012 Soil Candida spp.C. albicans CBS 2730 Gift from R. Rüchel (laboratory variant) C.glabrata CDC Y65 CDC C. krusei CDC 259-75 CDC C. parapsilosis CDC 22019CDC C. tropicalis CDC 38 CDC Fusarium spp. F. moniliforme ATCC 38159Human cutaneous infection F. oxysporum ATCC 4254 Unknown F. solani ATCC52628 Unknown Mucor spp. M. circinelloides ATCC 1209B Minus strain M.indicus ATCC 4857 Unknown M. plumbeus ATCC 4740 Unknown M. racemosusATCC 22365 Forest soil under basswood tree M. rouxii ATCC 24905 Ricefermentations Rhizopus spp. R. circinans ATCC 34101 Peach R. microsporusATCC 14050 Fatal human infection R. oryzae ATCC 34965 Pus from sinus(transplant recipient) R. stolonifer ATCC 14037 Zygospore germinationOther genera Apophysomyces elegans ATCC 46557 Human bronchial washingBlastomyces dermatitidis ATCC 60915 Attenuated mutant from humanNeosartorya fischeri ATCC 66781 Frozen pineapple juice concentratePseudallescheria boydii ATCC 36282 Human lung Sporothrix schenckii ATCC28184 Human arm lesion

TABLE 6 Specificity of P. marneffei probe versus DNAs from 13Penicillium species Species Mean absorbance_(650 nm) ± S.E. ^(a) P.marneffei 1.979 ± 0.087 (15) ^(b) P. camembertii 0.001 ± 0.001 (3) P.caseicolum 0.001 ± 0.001 (3) P. chrysogenum 0.020 ± 0.003 (3) P. glabrum0.017 ± 0.001 (3) P. griseofulvum 0.005 ± 0.002 (3) P. italicum 0.010 ±0.001 (3) P. janthinellum 0.009 ± 0.004 (3) P. purpurescens 0.005 ±0.002 (3) P. purpurogenum 0.001 ± 0.001 (3) P. roquefortii 0.004 ± 0.003(3) P. rubefaciens 0.020 ± 0.001 (3) P. spinulosum 0.001 ± 0.000 (3)^(a) Each sample was prepared and tested in at least three separateexperiments. The P. marneffei probe hybridized to its homologous speciesDNA but not to heterologous species DNA (P < 0.001). Mean absorbance_(650 nm) ± S.E. for all non-P. marneffei Penicillium species = 0.008 ±0.007 (n = 36). Negative control water blank was subtracted from allsamples.^(b) Mean absorbance _(650 nm) ± S.E. for 5 strains of P. marneffeitested in three separate experiments.

TABLE 7 Specificity of P. marneffei probe versus DNAs fromnon-Penicillium genera Mean absorbance _(650 nm) ± S.E.^(a) Probes for:Target DNA ^(b) P. marneffei Aspergillus spp Fusarium spp Mucor sppRhizopus spp P. marneffei 1.24 ± 0.12  0 ^(c) 0 0 0 (n = 3) Aspergillusspp. 0 1.67 ± 0.20 0 0 0 (n= 5) Fusarium spp. 0 0 1.28 ± 0.31 0 0 (n =3) Mucor spp. 0 0 0 1.85 ± 0.10 0 (n = 5) Rhizopus spp. 0 0 0 0 1.64 ±.33 (n = 4) All other fungi 0 0 0 0 0 (n = 10)^(a) Each sample was prepared and tested in at least two separateexperiments. Each probe specifically hybridized to its homologous DNAbut not to heterologous DNA (P < 0.001). Negative control# water blank was subtracted from all samples.^(b) Target DNA was derived from the number of different isolates (P.marneffei) or species in parentheses; species included those# from Mucor (M. circinelloides f circinelloides, M. indicus, M.plumbeus, M. racemosus, and M. rouxii), Rhizopus (R. circinans, R.microsporus, # and R. oryzae), Aspergillus (A. flavus, A. fumigatus, A.nidulans, A. niger, and A. terreus), Fusarium (F. moniliforme, F.oxysporum, # and F. solani), and all other fungi (C. albicans, C.glabrata, C. krusei, C. parapsilosis, C. tropicalis, B. dermatitidis, P.boydii, S. schenckii, N. fischeri, # and A. elegans).^(c) A value of zero was assigned for all mean absorbance values of lessthan 0.01 for ease of presentation.

TABLE 8 Specificity of Penicillium species probes versus DNAs from otherfungi Mean absorbance_(650 nm) ± S.E. ^(a) Probes for: Target DNA P.marneffei P. purpurogenum P. citrinum P. marneffei 1.635 ± 0.021   0^(b) 0 P. purpurogenum 0 0.434 ± 0.009 0 P. camembertii 0 0.198 ± 0.0050 P. citrinum 0 0 0.846 ±0.001 P. caseicolum 0 0 0 P. chrysogenum 0 0 0P. glabrum 0 0 0 P. griseofulvum 0 0 0 P. italicum 0 0 0 P. purpurescens0 0 0 P. roquefortii 0 0 0 A. fumigatus 0 0 0 C. albicans 0 0 0 F.solani 0 0 0 M. racemosus 0 0 0 P. boydii 0 0 0 S. schenckii 0 0 0^(a) Each sample was prepared and tested in at least two separateexperiments. Each probe hybridized to homologous DNA but not toheterologous DNA (P < 0.001) with the exception of the P. purpurogenumprobe with P. camembertii DNA. Negative control water blank wassubtracted from all samples.^(b) A value of zero was assigned for all mean absorbance values of lessthan 0.03 for ease of presentation.

Having illustrated and described the principals of the invention byseveral embodiments, it should be apparent that those embodiments can bemodified in arrangement and detail without departing from the principlesof the invention. Thus, the invention includes all such embodiments andvariations thereof, and their equivalents.

1. A method of detecting a dimorphic fungus, comprising: detectinghybridization between a dimorphic probe and an internal transcribedspacer-2 (ITS2) nucleic acid sequence of a dimorphic fungus within asample, wherein the dimorphic probe is capable of hybridizing to aregion comprising nucleotides 1-100 of the ITS2 nucleic acid sequence,and wherein the presence of hybridization indicates the presence of adimorphic fungus in the sample.
 2. The method according to claim 1,wherein the dimorphic fungus is Histoplasma capsulatum, Blastomycesdermatitidis, Coccidioides immitis, Penicillium marneffei, orParacoccidioides brasiliensis or a hybrid thereof.
 3. The method ofclaim 1, further comprising amplifying the ITS2 nucleic acid sequencefrom the sample, thereby generating an amplified ITS2 sequence.
 4. Themethod of claim 3, wherein the ITS2 nucleic acid sequence is amplifiedby polymerase chain reaction or polymerase chain reaction-enzymeimmunoassay.
 5. The method of claim 4, wherein a primer consistingessentially of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 is used inthe polymerase chain reaction or the polymerase chain reaction-enzymeimmunoassay.
 6. (canceled)
 7. The method of claim 52, wherein thespecies-specific probe or the microbe-specific probe comprises at least15 contiguous nucleotides of SEQ ID NO: 5, SEQ ID NO:_(—)6, SEQ ID NO:7, or SEQ ID NO:
 8. 8. The method of claim 7, wherein the probecomprises at least 20 contiguous nucleotides of SEQ ID NO: 5, SEQ IDNO:_(—)6, or SEQ ID NO:
 7. 9. The method of claim 8, wherein thespecies-specific probe or the microbe-specific probe consistsessentially of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO:8.
 10. The method of claim 1, wherein the probe comprises at least 15contiguous nucleotides of SEQ ID NO:
 4. 11. The method according toclaim 10, wherein the probe comprises at least 20 contiguous nucleotidesof SEQ ID NO:
 4. 12. The method according to claim 11, wherein the probeconsists essentially of SEQ ID NO:
 4. 13. The method of claim 1, whereinthe dimorphic probe is capable of hybridizing to a region comprising afirst half of the ITS2 nucleic acid sequence.
 14. The method of claim 1,wherein the dimorphic probe is capable of hybridizing to a regioncomprising nucleotides 1-90 of the ITS2 sequence.
 15. The method ofclaim 1, wherein the dimorphic probe is capable of hybridizing to aregion comprising nucleotides 50-100 of the ITS2 sequence.
 16. Themethod of claim 1, wherein the dimorphic probe hybridizes to a portionof the ITS2 sequence consisting of from about nucleotide 40 to aboutnucleotide 70 of the ITS2 sequence.
 17. A method of differentiating adimorphic fungus from a non-dimorphic fungus, comprising: contacting asample with a dimorphic probe, wherein the dimorphic probe hybridizes toa region comprising nucleotides 1-200 of an internal transcribedspacer-2 (ITS2) nucleic acid sequence; and detecting a hybridizationbetween the region comprising nucleotides 1-200 of the ITS2 nucleic acidsequence and the dimorphic probe, wherein the detection of thehybridization indicates the presence of a dimorphic fungus within thesample.
 18. The method according to claim 17, wherein the dimorphicfungus is Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioidesimmitis, or Paracoccidioides brasiliensis.
 19. The method according toclaim 17, further comprising amplifying the ITS2 nucleic acid sequence.20. The method according to claim 55, wherein the species-specific probeor the microbe-specific probe comprises at least 15 contiguousnucleotides of SEQ ID NO: 5, SEQ ID NO:_(—)6, SEQ ID NO: 7, or SEQ IDNO:
 8. 21. The method according to claim 20, wherein thespecies-specific probe or the microbe-specific probe comprises at least20 contiguous nucleotides of SEQ ID NO: 5, SEQ ID NO:_(—)6, SEQ ID NO:7, or SEQ ID NO:
 8. 22. (canceled)
 23. The method of claim 17, whereinthe dimorphic probe comprises at least 15 contiguous nucleotides of SEQID NO:
 4. 24. The method according to claim 23, wherein the dimorphicprobe comprises at least 20 contiguous nucleotides of SEQ ID NO:
 4. 25.The method according to claim 24, wherein the dimorphic probe consistsessentially of SEQ ID NO:
 4. 26. The method according to claim 17,wherein the method is capable of differentiating the presence ofHistoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis,or Paracoccidioides brasiliensis, apart from the presence or absence ofSporothrix schenckii, Cryptococcus neoformans, a Candida species, orPneumocystis carinii.
 27. A kit for detecting the presence of adimorphic fungus in a biological sample, comprising: a dimorphic probecomprising at least 15 contiguous nucleotides of SEQ ID NO:; andinstructions for hybridizing the probe to an internal transcribedspacer-2 (ITS2) nucleic acid sequence of a dimorphic fungus within thebiological sample.
 28. The kit according to claim 27, further comprisinga primer consisting essentially of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ IDNO:3.
 29. The kit according to claim 27, wherein the dimorphic probe iscapable of hybridizing with an ITS2 nucleic acid of at least two ofHistoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis,or Paracoccidioides brasiliensis.
 30. An array for screening a samplefor the presence of or contamination by a dimorphic fungus, comprising:a plurality of dimorphic probes, wherein the dimorphic probes arecomplementary to a region comprising nucleotides 1-100 of an internaltranscribed spacer-2 (ITS2) nucleic acid sequence of a dimorphic fungus;and a substrate, wherein the plurality of dimorphic probes are arrayedon the substrate.
 31. The array according to claim 30, wherein theprobes are capable of hybridizing to the ITS2 nucleic acid sequence ofthe dimorphic fungus, and wherein the hybridization indicates that thenucleic acid originated from Histoplasma capsulatum, Blastomycesdermatitidis, Coccidioides immitis, or Paracoccidioides brasiliensis.32. The array according to claim 30, wherein the array is a microarray.33. The array of claim 30, wherein at least one dimorphic probecomprises at least 15 contiguous nucleotides of SEQ ID NO:.
 34. Thearray of claim 33, wherein the at least one dimorphic probe comprises atleast 20 contiguous nucleotides of SEQ ID NO:.
 35. The array accordingto claim 34, wherein the at least one dimorphic probe consistsessentially of SEQ ID NO:.
 36. A method of detecting Penicilliummarneffei, comprising: contacting a sample containing an internaltranscribed spacer-2 (ITS2) nucleic acid sequence of a Penicilliummarneffei with a probe; and detecting a hybridization between the ITS2nucleic acid sequence and the probe, wherein the detection of thehybridization indicates the presence of the Penicillium marneffei ITS2nucleic acid within the sample.
 37. The method according to claim 36,wherein the method is capable of differentiating the Penicilliummarneffei ITS2 nucleic acid is differentiated from an ITS2 nucleic acidof a second fungus.
 38. The method according to claim 37, wherein thesecond fungus is Penicillium camembertii, Penicillium caseicolum,Penicillium chrysogenum, Penicillium glabrum, Penicillium griseofulvum,Penicillium italicum, Penicillium janthinellum, Penicilliumpurpurescens, Penicillium citrinum, Penicillium purpurogenum,Penicillium roquefortii, Penicillium rubefaciens, Penicilliumspinulosum, Sporothrix schenckii, Cryptococcus neoformans, anAspergillus species, a Candida species, a Fusarium species, a Mucorspecies, a Rhizopus species, or Pneumocystis carinii.
 39. The methodaccording to claim 37, wherein the probe comprises at least 15contiguous nucleotides of SEQ ID NO: 4 or SEQ ID NO:
 10. 40. The methodaccording to claim 37, wherein the probe comprises at least 20contiguous nucleotides of SEQ ID NO: 4 or SEQ ID NO:
 10. 41. The methodaccording to claim 37, wherein the probe consists essentially of SEQ IDNO: 4 or SEQ ID NO:
 10. 42. A method of differentiating a dimorphicfungus from a non-dimorphic fungus, comprising: contacting a samplecontaining a nucleic acid of dimorphic fungus with a dimorphic probe,wherein the dimorphic fungus is Histoplasma capsulatum, Blastomycesdermatitidis, Coccidioides immitis, or Paracoccidioides brasiliensis;and detecting a hybridization between the nucleic acid and the probe,wherein the detection of the hybridization indicates the presence of thenucleic acid of the dimorphic fungus, and wherein the method is capableof differentiating the nucleic acid of the dimorphic fungus apart from anucleic acid of Sporothrix schenckii, Cryptococcus neoformans, a Candidaspecies, or Pneumocystis carinii.
 43. A kit for detecting the presenceof a dimorphic fungus in a biological sample, comprising: a dimorphicprobe capable of hybridizing to a region comprising nucleotides 1-100 ofan internal transcribed spacer-2 (ITS2) nucleic acid sequence; andinstructions for hybridizing the dimorphic probe to the ITS2 nucleicacid sequence.
 44. The kit of claim 43, further comprising a primer foramplifying the ITS2 nucleic acid sequence.
 45. The kit of claim 27,further comprising a species-specific probe or a microbe-specific probe.46. The kit of claim 45, wherein the species-specific probe or themicrobe-specific probe, comprises SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO:7, or SEQ ID NO:
 8. 47. The array of claim 30, further comprising aspecies-specific probe or a microbe-specific probe.
 48. The array ofclaim 47, wherein the species-specific probe or the microbe-specificprobe comprises SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO:8.
 49. The method of claim 1, wherein detecting hybridization comprisesdetecting a label on the dimorphic probe.
 50. The method of claim 49,wherein the label comprises a radioactive isotope, enzyme substrate,co-factor, ligand, chemiluminescent agent, fluorescent agent, hapten, orenzyme.
 51. The method of claim 1, further comprising: isolating DNAfrom the sample; and contacting the dimorphic probe with the DNA. 52.The method of claim 1, further comprising: detecting a hybridizationbetween a species-specific probe or a microbe-specific probe and theITS2 nucleic acid sequence of a dimorphic fungus within a sample. 53.The method of claim 17, wherein detecting hybridization comprisesdetecting a label on the dimorphic probe.
 54. The method of claim 53,wherein the label comprises a radioactive isotope, enzyme substrate,co-factor, ligand, chemiluminescent agent, fluorescent agent, hapten, orenzyme.
 55. The method of claim 17, further comprising: detecting ahybridization between a species-specific probe or a microbe-specificprobe and a ITS2 nucleic acid sequence present in the dimorphic fungus.56. The method of claim 17, further comprising: isolating DNA from thesample, thereby generating isolated DNA, wherein contacting the samplewith the dimorphic probe comprises contacting the dimorphic probe withthe isolated DNA.
 57. The method of claim 1, wherein the sample is abiological or environmental sample.
 58. The method of claim 17, whereinthe sample is a biological or environmental sample.
 59. A method forextracting DNA comprising: disrupting a cell with a granular material;precipitating cellular proteins, thereby generating a supernatant;incubating the supernatant with RNase; and collecting remaining DNA. 60.The method of claim 60, wherein the granular material is differentiatedby size ratio.